Serum-free mammalian cell culture medium, and uses thereof

ABSTRACT

The present invention provides a cell culture medium formulation that supports the in vitro cultivation, particularly in suspension, of mammalian cells, particularly epithelial cells and fibroblast cells, and methods for cultivating mammalian cells in suspension in vitro using these media. The media comprise a basal medium and a polyanionic or polyanionic compound, preferably a polysulfonated or polysulfated compound, and more preferably dextran sulfate. The present invention also provides chemically defined, protein-free eukaryotic cell culture media comprising an iron chelate and zinc, which is capable of supporting the growth (and particularly the high-density growth of mammalian cells) in suspension culture, increasing the level of expression of recombinant protein in cultured cells, and/or increasing virus production in cultured cells.

FIELD OF THE INVENTION

The present invention relates generally to cell culture mediumformulations. Specifically, the present invention provides serum-free,low-protein or protein-free, defined cell culture medium formulationsthat facilitate the in vitro cultivation of mammalian cells insuspension. The culture media of the present invention are particularlysuitable for suspension culture of epithelial cells, such as 293 humanembryonic kidney cells, and fibroblast cells, such as Chinese hamsterovary (CHO) cells.

BACKGROUND OF THE INVENTION Cell Culture Media

The requirements of mammalian cell culture in vitro comprise, inaddition to basic nutritional substances, a complex series of growthfactors (Werner, R. G. et al., Mammalian Cell Cultures Part I:Characterization, morphology and metabolism, in: Arzneim.-Forsch./DrugRes. 43:1134-1139 (1993)). Usually, these are added to the culturemedium by supplying it with animal sera or protein-fractions from animalsources. However, these chemically non-defined mixtures exhibit variablelot to lot composition. Such mixtures also represent a potential sourceof contaminants, including viruses and mycoplasmas. For production on anindustrial scale, the high price of the supplements and difficulties indownstream processing are additional considerations.

Cell culture media provide the nutrients necessary to maintain and growcells in a controlled, artificial and in vitro environment.Characteristics and compositions of the cell culture media varydepending on the particular cellular requirements. Important parametersinclude osmolarity, pH, and nutrient formulations.

Media formulations have been used to cultivate a number of cell typesincluding animal, plant and bacterial cells. Cells cultivated in culturemedia catabolize available nutrients and produce useful biologicalsubstances such as virus, monoclonal antibodies, hormones, growthfactors and the like. Such products have therapeutic applications and,with the advent of recombinant DNA technology, cells can be engineeredto produce large quantities of many of these products. Thus, the abilityto cultivate cells in vitro is not only important for the study of cellphysiology, but is also necessary for the production of usefulsubstances which may not otherwise be obtained by cost-effective means.

Cell culture media formulations have been well documented in theliterature and a number of media are commercially available. In earlycell culture work, media formulations were based upon the chemicalcomposition and physicochemical properties (e.g., osmolality, pH, etc.)of blood and were referred to as “physiological solutions” (Ringer, S.,J. Physiol. 3:380-393 (1880); Waymouth, C., In: Cells and Tissues inCulture, Vol. 1, Academic Press, London, pp. 99-142 (1965); Waymouth,C., In Vitro 6:109-127 (1970)). However, cells in different tissues ofthe mammalian body are exposed to different microenvironments withrespect to oxygen/carbon dioxide partial pressure and concentrations ofnutrients, vitamins, and trace elements; accordingly, successful invitro culture of different cell types will often require the use ofdifferent media formulations. Typical components of cell culture mediainclude amino acids, organic and inorganic salts, vitamins, tracemetals, sugars, lipids and nucleic acids, the types and amounts of whichmay vary depending upon the particular requirements of a given cell ortissue type.

Typically, cell culture media formulations are supplemented with a rangeof additives, including undefined components such as fetal bovine serum(FBS) (10-20% v/v) or extracts from animal embryos, organs or glands(0.5-10% v/v). While FBS is the most commonly applied supplement inanimal cell culture media, other serum sources are also routinely used,including newborn calf, horse and human. Organs or glands that have beenused to prepare extracts for the supplementation of culture mediainclude submaxillary gland (Cohen, S., J. Biol. Chem. 237:1555-1565(1961)), pituitary (Peehl, D. M., and Ham, R. G., In Vitro 16:516-525(1980); U.S. Pat. No. 4,673,649), hypothalamus (Maciag, T., et al.,Proc. Natl. Acad. Sci. USA 76:5674-5678 (1979); Gilchrest, B. A., etal., J. Cell. Physiol. 120:377-383 (1984)), ocular retina (Barretault,D., et al., Differentiation 18:29-42 (1981)) and brain (Maciag, T., etal., Science 211:1452-1454 (1981)). These types of chemically undefinedsupplements serve several useful functions in cell culture media(Lambert, K. J. et al., In: Animal Cell Biotechnology, Vol. 1, Spier, R.E. et al., Eds., Academic Press New York, pp. 85-122 (1985)). Forexample, these supplements provide carriers or chelators for labile orwater-insoluble nutrients; bind and neutralize toxic moieties; providehormones and growth factors, protease inhibitors and essential, oftenunidentified or undefined low molecular weight nutrients; and protectcells from physical stress and damage. Thus, serum or organ/glandextracts are commonly used as relatively low-cost supplements to providean optimal culture medium for the cultivation of animal cells.

Unfortunately, the use of serum or organ/gland extracts in tissueculture applications has several drawbacks (Lambert, K. J. et al., In:Animal Cell Biotechnology, Vol 1, Spier, R. E. et al., Eds., AcademicPress New York, pp. 85-122 (1985)). For example, the chemicalcompositions of these supplements and sera vary between lots, even froma single manufacturer. The supplements may also be contaminated withinfectious agents (e.g., mycoplasma and viruses) which can seriouslyundermine the health of the cultured cells and the quality of the finalproduct. The use of undefined components such as serum or animalextracts also prevents the true definition and elucidation of thenutritional and hormonal requirements of the cultured cells, thuseliminating the ability to study, in a controlled way, the effect ofspecific growth factors or nutrients on cell growth and differentiationin culture. Moreover, undefined supplements prevent the researcher fromstudying aberrant growth and differentiation and the disease-relatedchanges in cultured cells. Finally and most importantly to thoseemploying cell culture media in the industrial production of biologicalsubstances, serum and organ/gland extract supplementation of culturemedia can complicate and increase the costs of the purification of thedesired substances from the culture media due to nonspecificco-purification of serum or extract proteins.

Defined Media

Improved levels of recombinant protein expression are obtained fromcells grown in serum-free medium, relative to the level of expressionseen in cells grown in medium supplemented with serum (Battista, P. J.et al., Am. Biotech. Lab. 12:64-68 (1994)). However, serum-free mediamay still contain one or more of a variety of animal-derived components,including albumin, fetuin, various hormones and other proteins. Thepresence of proteins or peptides makes purification of recombinantprotein difficult, time-consuming, and expensive.

To overcome these drawbacks of the use of serum or organ/gland extracts,a number of so-called “defined” media have been developed. These media,which often are specifically formulated to support the culture of asingle cell type, contain no undefined supplements and insteadincorporate defined quantities of purified growth factors, proteins,lipoproteins and other substances usually provided by the serum orextract supplement. Since the components (and concentrations thereof) insuch culture media are precisely known, these media are generallyreferred to as “defined culture media.” Often used interchangeably with“defined culture media” is the term “serum-free media” or “SFM.” Anumber of SFM formulations are commercially available, such as thosedesigned to support the culture of endothelial cells, keratinocytes,monocytes/macrophages, lymphocytes, hematopoietic stem cells,fibroblasts, chondrocytes or hepatocytes which are available from LifeTechnologies, Inc. (Rockville, Md.). The distinction between SFM anddefined media, however, is that SFM are media devoid of serum andprotein fractions (e.g., serum albumin), but not necessarily of otherundefined components such as organ/gland extracts. Indeed, several SFMthat have been reported or that are available commercially contain suchundefined components, including several formulations supporting in vitroculture of keratinocytes (Boyce, S. T., and Ham, R. G., J. Invest.Dermatol. 81:33 (1983); Wille, J. J., et al., J. Cell. Physiol. 121:31(1984); Pittelkow, M. R., and Scott, R. E., Mayo Clin. Proc. 61:771(1986); Pirisi, L., et al., J. Virol. 61:1061 (1987); Shipley, G. D.,and Pittelkow, M. R., Arch. Dermatol. 123:1541 (1987); Shipley, G. D.,et al., J. Cell. Physiol. 138:511-518 (1989); Daley, J. P., et al.,FOCUS (GIBCO/LTI) 12:68 (1990); U.S. Pat. Nos. 4,673,649 and 4,940,666).SFM thus cannot be considered to be defined media in the true definitionof the term.

Defined media generally provide several distinct advantages to the user.For example, the use of defined media facilitates the investigation ofthe effects of a specific growth factor or other medium component oncellular physiology, which may be masked when the cells are cultivatedin serum- or extract-containing media. In addition, defined mediatypically contain much lower quantities of protein (indeed, definedmedia are often termed “low protein media”) than those containing serumor extracts, rendering purification of biological substances produced bycells cultured in defined media far simpler and more cost-effective.

Some extremely simple defined media, which consist essentially ofvitamins, amino acids, organic and inorganic salts and buffers have beenused for cell culture. Such media (often called “basal media”), however,are usually seriously deficient in the nutritional content required bymost animal cells. Accordingly, most defined media incorporate into thebasal media additional components to make the media more nutritionallycomplex, but to maintain the serum-free and low protein content of themedia. Examples of such components include serum albumin from bovine(BSA) or human (HSA); certain growth factors derived from natural(animal) or recombinant sources such as EGF or FGF; lipids such as fattyacids, sterols and phospholipids; lipid derivatives and complexes suchas phosphoethanolamine, ethanolamine and lipoproteins; protein andsteroid hormones such as insulin, hydrocortisone and progesterone;nucleotide precursors; and certain trace elements (reviewed by Waymouth,C., in: Cell Culture Methods for Molecular and Cell Biology, Vol. 1:Methods for Preparation of Media, Supplements, and Substrata forSerum-Free Animal Cell Culture, Barnes, D. W., et al., eds., New York:Alan R. Liss, Inc., pp. 23-68 (1984), and by Gospodarowicz, D., Id., atpp 69-86 (1984)).

The use of animal protein supplements in cell culture media, however,also has certain drawbacks. For example, there is a risk that theculture medium and/or products purified from it may be immunogenic,particularly if the supplements are derived from an animal differentfrom the source of the cells to be cultured. If biological substances tobe used as therapeutics are purified from such culture media, certainamounts of these immunogenic proteins or peptides may be co-purified andmay induce an immunological reaction, up to and including anaphylaxis,in an animal receiving such therapeutics.

To obviate this potential problem, supplements derived from the samespecies as the cells to be cultured may be used. For example, culture ofhuman cells may be facilitated using HSA as a supplement, while mediafor the culture of bovine cells would instead use BSA. This approach,however, runs the risks of introducing contaminants and adventitiouspathogens into the culture medium (such as Creutzfeld-Jakob Disease(CJD) from HSA preparations, or Bovine Spongiform Encephalopathy (“MadCow Disease”) virus from BSA preparations), which can obviouslynegatively impact the use of such media in the preparation of animal andhuman therapeutics. In fact, for such safety reasons, the biotechnologyindustry and government agencies are increasingly regulating,discouraging and even forbidding the use of cell culture mediacontaining animal-derived proteins which may contain such pathogens.

Non-Animal Peptide Supplements

To overcome the limitations of the use of animal proteins in SFM,several attempts have been made to construct animal cell culture mediathat are completely free of animal proteins. For example, some culturemedia have incorporated extracts of yeast cells into the basal medium(see, for example, U.K. Patent Application No. GB 901673; Keay, L.,Biotechnol. Bioengin. 17:745-764 (1975)) to provide sources of nitrogenand other essential nutrients. In another approach, hydrolysates ofwheat gluten have been used, with or without addition of yeast extract,to promote in vitro growth of animal cells (Japanese Patent ApplicationNo. JP 2-49579). Still other media have been developed in which serum isreplaced by enzymatic digests of meat, or of proteins such asα-lactalbumin or casein (e.g., peptone), which have been traditionallyused in bacterial culture (Lasfargues, E. Y., et al., In Vitro8(6):494-500 (1973); Keay, L., Biotechnol. Bioeng. 17:745-764 (1975);Keay, L., Biotechnol. Bioeng. 19:399-411 (1977); Schlager, E.-J., J.Immunol. Meth. 194:191-199 (1996)). None of these approaches, however,provided a culture medium optimal for the cultivation of a variety ofanimal cells. Moreover, extracts from certain plants, including wheat,barley, rye and oats have been shown to inhibit protein synthesis incell-free systems derived from animal cells (Coleman, W. H., andRoberts, W. K., Biochim. Biophys. Acta 696:239-244 (1982)), suggestingthat the use of peptides derived from these plants in cell culture mediamay actually inhibit, rather than stimulate, the growth of animal cellsin vitro. More recently, animal cell culture SFM formulations comprisingrice peptides have been described and shown to be useful in cultivationof a variety of normal and transformed animal cells (see co-pending,commonly owned U.S. Application No. 60/028,197, filed Oct. 10, 1996, thedisclosure of which is incorporated herein by reference in itsentirety).

Epithelial Cells

Overview

The epithelium lines the internal and external surfaces of the organsand glands of higher organisms. Because of this localization at theexternal interface between the environment and the organism (e.g., theskin) or at the internal interface between an organ and the interstitialspace (e.g., the intestinal mucosal lining), the epithelium has a majorrole in the maintenance of homeostasis. The epithelium carries out thisfunction, for example, by regulating transport and permeability ofnutrients and wastes (Freshney, R. I., in: Culture of Epithelial Cells,Freshney, R. I., ed., New York: Wiley-Liss, pp. 1-23 (1992)).

The cells making up the epithelium are generically termed epithelialcells. These cells may be present in multiple layers as in the skin, orin a single layer as in the lung alveoli. As might be expected, thestructure, function and physiology of epithelial cells are oftentissue-specific. For example, the epidermal epithelial cells of the skinare organized as stratified squamous epithelium and are primarilyinvolved in forming a protective barrier for the organism, while thesecretory epithelial cells of many glands are often found in singlelayers of cuboidal cells that have a major role in producing secretoryproteins and glycoproteins. Regardless of their location or function,however, epithelial cells are usually regenerative. That is, undernormal conditions, or in response to injury or other activatingstimulus, epithelial cells are capable of dividing or growing. Thisregenerative capacity has facilitated the in vitro manipulation ofepithelial cells, to the point where a variety of primary epithelialcells and cell lines have been successfully cultivated in vitro(Freshney, Id.).

293 Cells

While the isolation and use of a variety of epithelial cells andepithelial cell lines have been reported in the literature, the humanembryonic kidney cell line 293 (“293 cells”), which exhibits epithelialmorphology, has proven particularly useful for studies of the expressionof exogenous ligand receptors, production of viruses and expression ofallogeneic and xenogeneic recombinant proteins. For example, U.S. Pat.No. 5,166,066 describes the construction of a stable 293 cell linecomprising functional GABA receptors that include a benzodiazepinebinding site, that have proven useful in identification and screening ofcandidate psychoactive drugs. 293 cells have also been used to produceviruses such as natural and recombinant adenoviruses (Garnier, A., etal., Cytotechnol. 15:145-155 (1994); Bout, A., et al., Cancer GeneTherapy 3(6):S24, abs. P-52 (1996); Wang, J.-W., et al., Cancer GeneTherapy 3(6):S24, abs. P-53 (1996)), which may be used for vaccineproduction or construction of adenovirus vectors for recombinant proteinexpression. Finally, 293 cells have proven useful in large-scaleproduction of a variety of recombinant human proteins (Berg, D. T., etal., BioTechniques 14(6):972-978 (1993); Peshwa, M. V., et al.,Biotechnol. Bioeng. 41:179-187 (1993); Garnier, A., et al., Cytotechnol.15:145-155 (1994)).

Fibroblast Cells

Overview

Cells loosely called fibroblasts have been isolated from many differenttissues and are understood to be connective tissue cells. It is clearlypossible to cultivate cell lines, loosely termed fibroblastic cells,from embryonic and adult tissues. Fibroblasts cells characteristicallyhave a “spindle” appearance. Fibroblast-like cells have morphologicalcharacteristics typical of fibroblast cells. Under a light microscopethe cells appear pointed and elongated (“spindle shaped”) when they growas a monolayer on the surface of a culture vessel. Cell lines can beregarded as fibroblast or fibroblast-like after confirmation withappropriate markers, such as collagen, type I ((Freshney, R. I., in:Culture of Epithelial Cells, Freshney, R. I., ed., New York: Wiley-Liss,pp. 1-23 (1987)).

CHO Cells

CHO cells have been classified as both epithelial and fibroblast cellsderived from the Chinese hamster ovary. A cell line started from Chinesehamster ovary (CHO-K1) (Kao, F.-T. And Puck, T. T., Proc. Natl. Acad.Sci. USA 60:1275-1281 (1968) has been in culture for many years but itsidentity is still not confirmed.

U.S. Pat. No. 5,316,938 discloses a medium for growing CHO cells insuspension which is essentially free of protein, lipid, and carbohydrateisolated from an animal source. This patent teaches that zinc is anoptional ingredient and that it is preferable to supplement the mediumwith recombinant insulin.

U.S. Pat. No. 5,122,469 discloses a protein-free medium whichfacilitates the expression of recombinant protein in CHO cells. Thispatent teaches that it is preferable to supplement the medium with bothinsulin and transferrin.

Zang, M. et al., Bio/Technology 13:389-392 (1995) discloses aprotein-free medium for growing CHO cells in suspension culture forrecombinant protein expression. See also U.S. Pat. Nos. 5,316,938 and5,122,469.

U.S. Pat. No. 4,767,704 discloses a protein-free medium whichfacilitates the long-term growth of antibody-producing monolayerhybridoma cells.

Suspension Cells

As noted above, most primary mammalian epithelial cells, mammalianfibroblast cells, epithelial cell lines, and fibroblast cell lines aretypically grown in monolayer culture. For some applications, however, itwould be advantageous to cultivate such cells as suspension cultures.For example, suspension cultures grow in a three-dimensional space.Monolayer cultures in similar-sized vessels, however, can only growtwo-dimensionally on the vessel surface. Thus, suspension culturestypically result in higher cell yields, and correspondingly higheryields of biologicals (e.g., viruses, recombinant polypeptides, etc.)compared to monolayer cultures. In addition, suspension cultures areoften easier to feed and scale-up, via simple addition of fresh culturemedia (dilution subculturing) to the culture vessel rather thantrypsinization and centrifugation as is often required with monolayercultures.

Many anchorage-dependent cells, such as primary epithelial cells,primary fibroblast cells, epithelial cell lines, and fibroblast celllines, however, are not easily adapted to suspension culture. Since theyare typically dependent upon anchorage to a substrate for optimalgrowth, growth of these cells in suspension may require their attachmentto microcarriers such as latex or collagen beads. Thus, cells grown inthis fashion, while capable of higher density culture than traditionalmonolayer cultures, are still technically attached to a surface;subculturing of these cells therefore requires similar steps as thosedescribed above for monolayer cultures. Furthermore, when large batch orfermenter cultures are established, a large volume of microcarriersoften settles to the bottom of the culture vessel, thereby requiring amore complicated agitation mechanism to keep the microcarriers (andthus, the cells) in suspension without causing shear damage to the cells(Peshwa, M. V., et al., Biotechnol. Bioeng. 41:179-187 (1993)).

Although many transformed cells are capable of being grown in suspension(Freshney, R. I., Culture of Animal Cells: A Manual of Basic Technique,New York: Alan R. Liss, Inc., pp. 123-125 (1983)), successful suspensioncultures often require relatively high-protein media or supplementationof the media with serum or serum components (such as the attachmentfactors fibronectin and/or vitronectin), or sophisticated perfusionculture control systems (Kyung, Y.-S., et al., Cytotechnol. 14:183-190(1994)), which may be disadvantageous for the reasons discussed above.In addition, many epithelial cells when grown in suspension formaggregates or “clumps” which may interfere with successful subculturingand reduce growth rate and production of biologicals by the cultures.When clumping occurs, the overall cellular surface area exposed tomedium is decreased and the cells are deprived of nutrition. As aresult, growth slows, diminished cell densities are obtained, andprotein expression is compromised.

Thus, there remains a need for a chemically defined, protein-free mediumwhich facilitates the growth of mammalian cells to high density and/orincreases the level of expression of recombinant protein, reduces cellclumping, and which does not require supplementation with animalproteins, such as transferrin and insulin.

There also remains a need remains for defined culture media, that areserum-free, and low-protein or protein-free, for the suspensioncultivation of mammalian cells that are normally anchorage-dependent,including epithelial cells and fibroblast cells, such as 293 cells andCHO cells. Such culture media will facilitate studies of the effects ofgrowth factors and other stimuli on cellular physiology, will alloweasier and more cost-effective production and purification of biologicalsubstances (e.g., viruses, recombinant proteins, etc.) produced bycultured mammalian cells in the biotechnology industry, and will providemore consistent results in methods employing the cultivation ofmammalian cells.

SUMMARY OF THE INVENTION

The present invention provides a method of cultivating a mammalian cellin suspension in vitro, the method comprising (a) obtaining a mammaliancell to be cultivated in suspension; and (b) contacting the cell with aserum-free cell culture medium comprising at least one polyanionic orpolycationic compound, wherein the medium supports the cultivation ofsaid cell in suspension. The present invention also relates to media forsuspension culture and to compositions comprising mammalian cells insuch suspension culture.

The present invention also relates to a method of replacing protein(particularly animal derived protein) in mammalian cell culture media.In particular, the invention relates to replacing transferrin and/orinsulin, to media containing such replacements, and to compositionscomprising mammalian cells in such media.

The present invention relates in particular to a medium referred toherein as the “suspension medium” and to a medium referred to herein asthe “replacement medium.”

The Suspension Medium

The present invention is directed to a serum-free cell culture mediumcomprising one or more polyanionic or polycationic compounds, whereinthe medium is capable of supporting the suspension cultivation of amammalian epithelial of fibroblast cells in vitro. In the suspensionmedium, the polyanionic compound is preferably a polysulfonated orpolysulfated compound, more preferably heparin, dextran sulfate, heparansulfate, dermatan sulfate, chondroitin sulfate, pentosan polysulfate, aproteoglycan or the like, and most preferably dextran sulfate, whichpreferably has a molecular weight of about 5,000 daltons.

In particular, the invention is directed to such culture media thatfurther comprise one or more ingredients selected from the group ofingredients consisting of one or more amino acids, one or more vitamins,one or more inorganic salts, one or more sugars, one or more bufferingsalts, one or more lipids, one or more insulins (or insulin substitutes)and one or more transferrins (or transferrin substitutes). The preferredsugar used in the media of the invention is D-glucose, while thepreferred buffer salt isN-[2-hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid] (HEPES). Theinvention is also directed to such culture media which may optionallycomprise one or more supplements selected from the group of supplementsconsisting of one or more cytokines, heparin, one or more animalpeptides, one or more yeast peptides and one or more plant peptides(which are preferably one or more rice peptides or one or more soypeptides). The amino acid ingredient of the present media preferablycomprises one or more amino acids selected from the group consisting ofL-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine,L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine,L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine and L-valine. The vitaminingredient of the present media preferably comprises one or morevitamins selected from the group consisting of biotin, choline chloride,D-Ca⁺⁺-pantothenate, folic acid, i-inositol, niacinamide, pyridoxine,riboflavin, thiamine and vitamin B₁₂. The inorganic salt ingredient ofthe present media preferably comprises one or more inorganic saltsselected from the group consisting of one or more calcium salts,Fe(NO₃)₃, KCl, one or more magnesium salts, one or more manganese salts,NaCl, NaHCO₃, Na₂HPO₄, one or more selenium salts, one or more vanadiumsalts and one or more zinc salts.

The invention is also directed to a cell culture medium comprising theingredients ethanolamine, D-glucose,N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES),insulin, linoleic acid, lipoic acid, phenol red, PLURONIC F68,putrescine, sodium pyruvate, transferrin, L-alanine, L-arginine,L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine,glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, L-valine, biotin, choline chloride, D-Ca⁺⁺-pantothenate,folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine,vitamin B₁₂, one or more calcium salts, Fe(NO₃)₃, KCl, one or moremagnesium salts, one or more manganese salts, NaCl, NaHCO₃, Na₂HPO₄, oneor more selenium salts, one or more vanadium salts and one or more zincsalts, wherein each ingredient is present in an amount which supportsthe suspension cultivation of a mammalian epithelial cell in vitro. Theinvention is also directed to such media which further comprise dextransulfate, and which optionally comprise one or more supplements selectedfrom the group consisting of one or more cytokines, heparin, one or moreanimal peptides, one or more yeast peptides and one or more plantpeptides (most preferably one or more rice peptides or one or more soypeptides).

The invention is also directed to a mammalian cell culture mediumobtained by combining a basal medium with dextran sulfate (whichpreferably has a molecular weight of about 5,000 daltons), wherein themedium is capable of supporting the suspension cultivation of amammalian epithelial or fibroblast cell in vitro. In one preferred suchmedium, the basal medium is obtained by combining one or moreingredients selected from the group consisting of ethanolamine,D-glucose, N-[2-hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid](HEPES), insulin, linoleic acid, lipoic acid, phenol red, PLURONIC F68,putrescine, sodium pyruvate, transferrin, L-alanine, L-arginine,L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine,glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, L-valine, biotin, choline chloride, D-Ca⁺⁺-pantothenate,folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine,vitamin B₁₂, one or more calcium salts, Fe(NO₃)₃, KCl, one or moremagnesium salts, one or more manganese salts, NaCl, NaHCO₃, Na₂HPO₄, oneor more selenium salts, one or more vanadium salts and one or more zincsalts, wherein each ingredient is added in an amount which supports thesuspension cultivation of a mammalian epithelial or fibroblast cell invitro. The invention is also directed to a medium obtained by combiningthe media obtained as described above and one or more supplementsselected from the group consisting of one or more cytokines, heparin,one or more animal peptides, one or more yeast peptides and one or moreplant peptides (preferably one or more rice peptides or one or more soypeptides).

The media provided by the present invention may be protein-free, and maybe a 1× formulation or concentrated as a 10× or higher formulation. Thebasal medium of the present invention comprises a number of ingredients,including amino acids, vitamins, organic and inorganic salts, sugars andother components, each ingredient being present in an amount whichsupports the cultivation of a mammalian epithelial cell in vitro. Themedium may be used to culture a variety of mammalian cells, includingprimary epithelial cells (e.g., keratinocytes, cervical epithelialcells, bronchial epithelial cells, tracheal epithelial cells, kidneyepithelial cells and retinal epithelial cells) and established celllines (e.g., 293 embryonic kidney cells, HeLa cervical epithelial cellsand PER-C6 retinal cells, MDBK (NBL-1) cells, CRFK cells, MDCK cells,CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells,HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells,NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13,2RA cells, WISH cells, BS-C-I cells, LLC-MK₂ cells, Clone M-3 cells,I-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK₁ cells, PK(15)cells, GH₁ cells, GH₃ cells, L2 cells, LLC-RC 256 cells, MH₁C₁ cells, XCcells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivativesthereof), fibroblast cells from any tissue or organ (including but notlimited to heart, liver, kidney, colon, intesting, esophagus, stomach,neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein,capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow,and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g.,CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells,citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl₁ cells, CHO cells,CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells,DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C₃H/IOTI/2 cells,HSDM₁C₃ cells, KLN205 cells, McCoy cells, Mouse L cells, Strain 2071(Mouse L) cells, L-M strain (Mouse L) cells, L-MTK⁻ (Mouse L) cells,NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indianmuntjac cells, SIRC cells, C_(II) cells, and Jensen cells, orderivatives thereof).

Cells supported by the medium of the present invention may be derivedfrom any animal, preferably a mammal, and most preferably a human. Thecells cultivated in the present media may be normal cells or abnormalcells (i.e., transformed cells, established cells, or cells derived fromdiseased tissue samples). The media of the invention may also beprepared in different forms, such as dry powder media (“DPM”), as liquidmedia or as media concentrates.

The present invention also provides methods of cultivating mammalianepithelial or fibroblast cells using the culture medium formulationsdisclosed herein, comprising (a) contacting the cells with the cellculture media of the invention; and (b) cultivating the cells underconditions suitable to support cultivation of the cells. Preferably,cells cultivated according to these methods (which may include any ofthe cells described above) are cultivated in suspension.

The invention also provides kits for use in the cultivation of amammalian epithelial cell. Kits according to the present inventioncomprise one or more containers, wherein a first container contains theculture medium of the invention. Additional kits of the inventioncomprise one or more containers wherein a first container contains abasal culture medium as described above and a second container containsone or more polyanionic or polycationic compounds, preferably apolysulfonated or polysulfated compound, more preferably heparin,dextran sulfate, heparan sulfate, dermatan sulfate, chondroitin sulfate,pentosan polysulfate, a proteoglycan or the like, and most preferablydextran sulfate which preferably has a molecular weight of about 5,000daltons. These kits may further comprise one or more additionalcontainers containing one or more supplements selected from the groupconsisting of one or more cytokines, heparin, one or more animalpeptides, one or more yeast peptides and one or more plant peptides(which are preferably one or more rice peptides or one or more soypeptides).

The invention further provides compositions comprising the culture mediaof the present invention, which optionally may further comprise one ormore mammalian epithelial or fibroblast cells, such as those describedabove, particularly one or more 293 embryonic kidney cells, PER-C6retinal cells, and CHO cells.

The present invention further relates to methods of cultivatingmammalian cells (particularly those described above and mostparticularly 293 embryonic kidney epithelial cells, PER-C6, and CHOcells) in suspension comprising (a) obtaining a mammalian cell to becultivated in suspension; and (b) contacting the cell with the culturemedia of the invention under conditions sufficient to support thecultivation of the cell in suspension.

The present invention further relates to methods of producing a virus,and to viruses produced by these methods, the methods comprising (a)obtaining a mammalian cell, preferably a mammalian cell described aboveand most preferably a 293 embryonic kidney epithelial cell, PER-C6, andCHO cells, to be infected with a virus; (b) contacting the cell with avirus under conditions suitable to promote the infection of the cell bythe virus; and (c) cultivating the cell in the culture medium of theinvention under conditions suitable to promote the production of thevirus by the cell. Viruses which may be produced according to thesemethods include adenoviruses, adeno-associated viruses and retroviruses.

The present invention further relates to methods of producing apolypeptide, and to polypeptides produced by these methods, the methodscomprising (a) obtaining a mammalian cell, preferably a mammalian celldescribed above and most preferably a 293 embryonic kidney epithelialcell, PER-C6, and CHO cell, that has been genetically engineered toproduce a polypeptide; and (b) cultivating the mammalian cell in theculture medium of the invention under conditions favoring the expressionof the desired polypeptide by the mammalian cell.

The Replacement Medium

The present invention also provides the replacement medium, a chemicallydefined, protein-free eukaryotic (e.g., mammalian) cell culture mediumcomprising a Fe²⁺ and/or Fe³⁺ chelate and/or a Zn²⁺ salt, and optionallyat least one polyanionic or polycationic compound as defined herein,which is capable of supporting the growth (in particular, thehigh-density growth of any of mentioned mammalian cells, andparticularly those described above, and preferably, CHO cells, PER-C6cells, and 293 cells) in suspension culture, increasing the level ofexpression of recombinant protein in cultured cells, and/or increasingvirus production in cultured cells. Further, the present inventionprovides a eukaryotic cell culture medium, obtained by combining an ironchelate and zinc, which is capable of supporting the density growth (inparticular, the high-density growth of any of mentioned mammalian cells,and particularly those described above, and preferably, CHO cells,PER-C6 cells, and 293 cells) in suspension culture, increasing the levelof expression of recombinant protein in cultured cells, and/orincreasing virus production in cultured cells. Further, the presentinvention provides a method of cultivating mammalian cells, andparticularly CHO cells, in suspension culture such that said cellsexpress a recombinant protein comprising the steps of contacting saidcells with the eukaryotic cell culture medium, wherein the iron chelateand zinc are present in an amount which supports the growth of mammaliancells in culture, and optionally together with a polyanionic orpolycationic compound in an amount effective to reduce clumping of thecells compared to when the compound is not added, and cultivating thecells under conditions suitable to support both the growth (inparticular, the high-density growth of any of mentioned mammalian cells,and particularly those described above, and preferably, CHO cells,PER-C6 cells, and 293 cells) in suspension culture, increasing the levelof expression of recombinant protein in cultured cells, and/orincreasing virus production in cultured cells.

The media provided by the present invention may be protein-free, and maybe a 1× formulation or concentrated as a 10× or higher formulation. Thebasal medium of the present invention comprises a number of ingredients,including amino acids, vitamins, organic and inorganic salts, sugars andother components, each ingredient being present in an amount whichsupports the cultivation of a mammalian epithelial cell in vitro. Themedium may be used to culture a variety of mammalian cells, includingprimary epithelial cells (e.g., keratinocytes, cervical epithelialcells, bronchial epithelial cells, tracheal epithelial cells, kidneyepithelial cells and retinal epithelial cells) and established celllines (e.g., 293 embryonic kidney cells, HeLa cervical epithelial cellsand PER-C6 retinal cells, MDBK (NBL-1) cells, CRFK cells, MDCK cells,CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells,HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells,NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13,2RA cells, WISH cells, BS-C-I cells, LLC-MK₂ cells, Clone M-3 cells,I-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK₁ cells, PK(15)cells, GH₁ cells, GH₃ cells, L2 cells, LLC-RC 256 cells, MH₁C₁ cells, XCcells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivativesthereof), fibroblast cells from any tissue or organ (including but notlimited to heart, liver, kidney, colon, intesting, esophagus, stomach,neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein,capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow,and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g.,CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells,citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl₁ cells, CHO cells,CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells,DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C₃H/IOTI/2 cells,HSDM₁C₃ cells, KLN205 cells, McCoy cells, Mouse L cells, Strain 2071(Mouse L) cells, L-M strain (Mouse L) cells, L-MTK⁻ (Mouse L) cells,NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indianmuntjac cells, SIRC cells, C_(II) cells, and Jensen cells, orderivatives thereof).

Cells supported by the medium of the present invention may be derivedfrom any animal, preferably a mammal, and most preferably a human. Thecells cultivated in the present media may be normal cells or abnormalcells (i.e., transformed cells, established cells, or cells derived fromdiseased tissue samples). The media of the invention may also beprepared in different forms, such as dry powder media (“DPM”), as liquidmedia or as media concentrates.

The present invention also provides methods of cultivating mammalianepithelial or fibroblast cells using the culture medium formulationsdisclosed herein, comprising (a) contacting the cells with the cellculture media of the invention; and (b) cultivating the cells underconditions suitable to support cultivation of the cells. Preferably,cells cultivated according to these methods (which may include any ofthe cells described above) are cultivated in suspension.

The medium of the present invention is a chemically defined formulationwhich contains no protein or hydrolysates of either plant or animalorigin. Although the invention is not bound by any particular theory, itis believed that the ability of the medium of the present invention tofacilitate the growth of mammalian cells is due to the replacement ofinsulin by zinc and/or the replacement of transferrin with an ironchelate. Moreover, when supplemented with dextran sulfate, the mediumfacilitates growth (in particular, the high-density growth of any ofmentioned mammalian cells, and particularly those described above, andpreferably, CHO cells, PER-C6 cells, and 293 cells) in suspensionculture, increases the level of expression of recombinant protein incultured cells, and/or increases virus production in cultured cellswithout clumping.

The medium of the present invention can be used to grow mammalian cells(in particular, to high-density) of any of mentioned mammalian cells,and particularly those described above, and preferably, CHO cells,PER-C6 cells, and 293 cells) to high density, to facilitate theexpression of recombinant protein in such cells, and/or to increasevirus production in cultured cells without clumping. The medium isadvantageous because it is chemically defined, it is protein free, andit does not require supplementation with transferrin, insulin, or otherproteins to facilitate cell growth and/or expression of recombinantprotein. In addition, the protein-free nature of the medium of thepresent invention greatly simplifies the purification of recombinantprotein.

The invention also provides kits for use in the cultivation of amammalian epithelial cell. Kits according to the present inventioncomprise one or more containers, wherein a first container contains theculture medium of the invention. Additional kits of the inventioncomprise one or more containers wherein a first container contains abasal culture medium as described above and a second container containsone or more polyanionic or polycationic compounds, preferably apolysulfonated or polysulfated compound, more preferably heparin,dextran sulfate, heparan sulfate, dermatan sulfate, chondroitin sulfate,pentosan polysulfate, a proteoglycan or the like, and most preferablydextran sulfate which preferably has a molecular weight of about 5,000daltons.

Other preferred embodiments of the present invention will be apparent toone of ordinary skill in light of the following drawings and descriptionof the invention, and of the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a line graph demonstrating cell growth (▪) and percentviable cells (), over a five-day time course, of 293 cells cultured insuspension in the suspension culture media of the invention.

FIG. 2 depicts a line graph demonstrating viable cell density, over aseven-day time course, of 293 cells cultured in suspension in thesuspension culture media without transferrin (), in the present culturemedia with human transferrin (▪), or in the present culture media inwhich transferrin was replaced with 60 μM ferric chloride/sodium citratechelate (▴) or with 40 μM ferrous sulfate/EDTA chelate (∇).

FIG. 3 depicts a bar graph demonstrating the production of adenovirus-5in 293 cells cultured in the suspension culture media of the inventionover a five-day time course. TCID50=tissue culture infectious dose−50%.

FIG. 4 depicts a line graph demonstrating β-galactosidase production, asa function of viable cell number over a six-day time course, by 293cells cultured in the suspension culture media of the invention.

FIG. 5A depicts a bar graph showing the effect oflow-protein/serum-free, essentially protein-free, andprotein-free/chemically defined media on the growth of rβ-gal CHO cells.FIG. 5B depicts a bar graph showing the effect of lowprotein/serum-free, essentially protein-free, andprotein-free/chemically defined media on the expression ofrβ-galactosidase in rβ-gal CHO cells.

FIG. 6A depicts a bar graph showing the effect of lowprotein/serum-free, essentially protein-free, and proteinfree/chemically defined media on the growth of rβ-gal CHO cells. In thisfigure, “g” refers to the growth phase and “p” refers to the productionphase. FIG. 6B depicts a bar graph showing the effect of lowprotein/serum-free, essentially protein-free, andprotein-free/chemically defined media on the expression ofrβ-galactosidase in rβ-gal CHO cells. In this figure, “g” refers to thegrowth phase and “p” refers to the production phase.

FIG. 7A depicts a bar graph showing the effect of methotrexate on thegrowth of rβ-gal CHO cells. FIG. 7B depicts a bar graph showing theeffect of methotrexate on the expression of rβ-galactosidase in rβ-galCHO cells.

FIG. 8A depicts a graph showing the effect of essentially protein-free() and protein-free/chemically defined (▪) media on rbGH CHO cells.FIG. 8B depicts a graph showing the effect of essentially protein-free() and protein-free/chemically defined (▪) media on rbGH expression inrbGH CHO cells.

FIG. 9A depicts a bar graph showing the effect of low-protein, insulin-and transferrin-containing medium and a protein-free/chemically definedmedium on the growth of rβ-gal CHO cells. FIG. 9B depicts a bar graphshowing the effect of low-protein, insulin- and transferrin-containingmedium and a protein-free/chemically defined medium on rβ-galactosidaseexpression in rβ-gal CHO cells.

FIG. 10 depicts a bar graph showing the effect of CD CHO, CHO III, andFMX-8 media on the growth of rβ-gal CHO cells.

FIG. 11 depicts a bar graph showing the effect of CD CHO, CHO III, andFMX-8 media on rβ-gal expression in rβ-gal CHO cells.

FIG. 12A shows the level of growth of rβ-gal CHO cells cultured in CDCHO medium in a shake flask (▪) and in a bioreactor (). FIG. 12B showsthe level of rβ-gal expression in cells cultured in CD CHO medium in ashake flask and in a bioreactor.

FIG. 13 depicts a bar graph showing the effect of dextran sulfate on thegrowth of rβ-gal CHO cells. In the figure, “A” is dextran sulfate (m.w.5,000) and “C” is dextran sulfate (m.w. 500,000).

FIG. 14 depicts a bar graph showing the effect of dextran sulfate onrβ-gal expression in rβ-gal CHO cells. In the figure, “A” is dextransulfate (m.w. 5,000) and “C” is dextran sulfate (m.w. 500,000).

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms conventionally usedin the field of cell culture media are utilized extensively. In order toprovide a clear and consistent understanding of the specification andclaims, and the scope to be given such terms, the following definitionsare provided.

The term “batch culture” refers to a culture allowed to progress frominoculation to conclusion without refeeding the cultured cells withfresh medium.

The term “cytokine” refers to a compound that induces a physiologicalresponse in a cell, such as growth, differentiation, senescence,apoptosis, cytotoxicity or antibody secretion. Included in thisdefinition of “cytokine” are growth factors, interleukins,colony-stimulating factors, interferons, lymphokines and the like.

By “cell culture” or “culture” is meant the maintenance of cells in anartificial, in vitro environment. It is to be understood, however, thatthe term “cell culture” is a generic term and may be used to encompassthe cultivation not only of individual cells, but also of tissues,organs, organ systems or whole organisms, for which the terms “tissueculture,” “organ culture,” “organ system culture” or “organotypicculture” may occasionally be used interchangeably with the term “cellculture.” The media of the present invention can be used to culture anyadherent mammalian cell (i.e., a cell which adheres to the culturevessel) and any mammalian cell which grows in suspension culture.

By “cultivation” is meant the maintenance of cells in vitro underconditions favoring growth, differentiation or continued viability, inan active or quiescent state, of the cells. In this sense, “cultivation”may be used interchangeably with “cell culture” or any of its synonymsdescribed above.

By “culture vessel” is meant a glass, plastic, or metal container thatcan provide an aseptic environment for culturing cells.

The phrases “cell culture medium,” “culture medium” (plural “media” ineach case) and “medium formulation” refer to a nutritive solution forcultivating cells and may be used interchangeably.

The term “contacting” refers to the placing of cells to be cultivated invitro into a culture vessel with the medium in which the cells are to becultivated. The term “contacting” encompasses mixing cells with medium,pipetting medium onto cells in a culture vessel, and submerging cells inculture medium.

The term “combining” refers to the mixing or admixing of ingredients ina cell culture medium formulation.

A “chemically defined” medium is one for which every ingredient isknown. A chemically defined medium is distinguished from serum,embryonic extracts, and hydrolysates, each of which contain unknowncomponents. The medium of the present invention is chemically definedand is free of proteins and peptides.

The term “high density” refers to a cellular density of about 1×10⁶ toabout 2×10⁷ cells/ml. In a preferred embodiment, the term refers to acellular density of about 1×10⁶ to about 5×10⁶ cells/ml in batchculture.

The term “ingredient” refers to any compound, whether of chemical orbiological origin, that can be used in cell culture media to maintain orpromote the growth of proliferation of cells. The terms “component,”“nutrient” and ingredient” can be used interchangeably and are all meantto refer to such compounds. Typical ingredients that are used in cellculture media include amino acids, salts, metals, sugars, lipids,nucleic acids, hormones, vitamins, fatty acids, proteins and the like.Other ingredients that promote or maintain cultivation of cells ex vivocan be selected by those of skill in the art, in accordance with theparticular need.

Each ingredient used in cell culture media has unique physical andchemical characteristics. By “compatible ingredients” is meant thosemedia nutrients which can be maintained in solution and form a “stable”combination. A solution containing “compatible ingredients” is said tobe “stable” when the ingredients do not degrade or decomposesubstantially into toxic compounds, or do not degrade or decomposesubstantially into compounds that can not be utilized or catabolized bythe cell culture. Ingredients are also considered “stable” ifdegradation can not be detected or when degradation occurs at a slowerrate when compared to decomposition of the same ingredient in a 1× cellculture media formulation. Glutamine, for example, in 1× mediaformulations, is known to degrade into pyrrolidone carboxylic acid andammonia. Glutamine in combination with divalent cations are considered“compatible ingredients” since little or no decomposition can bedetected over time. See U.S. Pat. No. 5,474,931.

Compatibility of media ingredients, in addition to stabilitymeasurements, are also determined by the “solubility” of the ingredientsin solution. The term “solubility” or “soluble” refers to the ability ofa ingredient to form a solution with other ingredients. Ingredients arethus compatible if they can be maintained in solution without forming ameasurable or detectable precipitate. Thus, the term “compatibleingredients” as used herein refers to the combination of particularculture media ingredients which, when mixed in solution either asconcentrated or 1× formulations, are “stable” and “soluble.”

A “protein-free” medium is one which contains no proteins or peptides. Aprotein-free medium is distinguished from low-protein and essentiallyprotein-free media, both of which contain proteins and/or peptides.

A cell culture medium is composed of a number of ingredients and theseingredients vary from one culture medium to another. A “1× formulation”is meant to refer to any aqueous solution that contains some or allingredients found in a cell culture medium at working concentrations.The “1× formulation” can refer to, for example, the cell culture mediumor to any subgroup of ingredients for that medium. The concentration ofan ingredient in a 1× solution is about the same as the concentration ofthat ingredient found in a cell culture formulation used for maintainingor cultivating cells in vitro. A cell culture medium used for the invitro cultivation of cells is a 1× formulation by definition. When anumber of ingredients are present, each ingredient in a 1× formulationhas a concentration about equal to the concentration of thoseingredients in a cell culture medium. For example, RPMI-1640 culturemedium contains, among other ingredients, 0.2 g/L L-arginine, 0.05 g/LL-asparagine, and 0.02 g/L L-aspartic acid. A “1× formulation” of theseamino acids contains about the same concentrations of these ingredientsin solution. Thus, when referring to a “1× formulation,” it is intendedthat each ingredient in solution has the same or about the sameconcentration as that found in the cell culture medium being described.The concentrations of ingredients in a 1× formulation of cell culturemedium are well known to those of ordinary skill in the art. See MethodsFor Preparation of Media, Supplements and Substrate For Serum-FreeAnimal Cell Culture, New York: Allen R. Liss (1984), which isincorporated by reference herein in its entirety. The osmolarity and/orpH, however, may differ in a 1× formulation compared to the culturemedium, particularly when fewer ingredients are contained in the 1×formulation.

A “10× formulation” is meant to refer to a solution wherein eachingredient in that solution is about 10 times more concentrated than thesame ingredient in the cell culture medium. For example, a 10×formulation of RPMI-1640 culture medium may contain, among otheringredients, 2.0 g/L L-arginine, 0.5 g/L L-asparagine, and 0.2 g/LL-aspartic acid (compare 1× formulation, above). A “10× formulation” maycontain a number of additional ingredients at a concentration about 10times that found in the 1× culture medium. As will be readily apparent,“25× formulation,” “50× formulation,” “100× formulation,” “500×formulation,” and “1000× formulation” designate solutions that containingredients at about 25-, 50-, 100-, 500-, or 1000-fold concentrations,respectively, as compared to a 1× cell culture medium. Again, theosmolarity and pH of the media formulation and concentrated solution mayvary.

Tissues, organs and organ systems derived from animals or constructed invitro or in vivo using methods routine in the art may similarly becultivated in the culture media of the present invention. Animals fromwhich cells can originate include human, monkey, ape, mouse, rat,hamster, rabbit, guinea pig, cow, swine, dog, horse, cat, goat, andsheep.

The media of the present invention can be used to grow mammalian cellsin suspension culture in bioreactors, roller bottles, and microcarriersystems.

Formulation of the Suspension Culture Media

The suspension media of the present invention is generally directed to aserum-free cell culture medium comprising one or more polyanionic orpolycationic compounds, wherein the medium is capable of supporting thesuspension cultivation of mammalian epithelial cells (epithelial orfibroblast) in vitro. In the present media, the polyanionic compound ispreferably a polysulfonated or polysulfated compound, more preferablyheparin, dextran sulfate, heparan sulfate, dermatan sulfate, chondroitinsulfate, pentosan polysulfate, a proteoglycan or the like, and mostpreferably dextran sulfate, which preferably has a molecular weight ofabout 5,000 daltons. The invention also relates generally to serum-freeculture media for use in suspension cultivation of a mammalian cell,comprising one or more of the above-described polyanionic orpolycationic compounds, particularly dextran sulfate. In addition, theinvention relates to serum-free culture media for use in producing avirus, the media comprising one or more of the above-describedpolyanionic or polycationic compounds, particularly dextran sulfate,wherein a virus-infected mammalian cell cultivated in suspension in themedia produces a higher virus titer than a mammalian cell not cultivatedin suspension in the media.

Basal and Complete Media

Any basal medium may be used in accordance with the present invention.Ingredients which the basal media of the present invention may includeare amino acids, vitamins, inorganic salts, sugars, buffering salts,lipids, insulin (or insulin substitute) and transferrin (or transferrinsubstitute). More specifically, the basal media may containethanolamine, D-glucose,N-[2-hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid] (HEPES),insulin, linoleic acid, lipoic acid, phenol red, PLURONIC F68,putrescine, sodium pyruvate. This culture medium contains no serum andtherefore is considered an SFM; it is also a low-protein medium sincethe only protein components are insulin and transferrin. Transferrin maybe in the iron-free form (i.e., apotransferrin) or in the iron-complexedform (i.e., ferrotransferrin or holotransferrin), and insulin, ifpresent, may be human- or animal-derived and may be natural orrecombinant. The medium may, of course, be made completely protein-freeby not including transferrin and insulin in the formulation. Transferrinmay be replaced by ferric citrate chelates at a concentration of about10-100 μM (preferably FeCl₃-sodium citrate chelate at about 60 μM) orferrous sulfate chelates at a concentration of about 10-100 μM(preferably FeSO₄EDTA chelate at about 40 μM). Insulin may be replacedby one or more zinc-containing compounds such alone or more zinc salts.Zinc-containing compounds which may be used include but are not limitedto ZnCl, Zn(NO₃)₂, ZnBr, and ZnSO₄, any of which may be present in theiranhydrous or hydrated (i.e., “.H₂O”) forms. Preferably, thezinc-containing compound used is ZnSO₄.7H₂O. In the protein-free mediumof the present invention, the concentration of zinc can be optimizedusing only routine experimentation. Typically, the concentration of zincin the 1× medium of the present invention may be about 0.07 to 0.73mg/L, and is preferably about 0354 mg/L. Each of these ingredients maybe obtained commercially, for example from Sigma (Saint Louis, Mo.).

Amino acid ingredients which may be included in the media of the presentinvention include L-alanine, L-arginine, L-asparagine, L-aspartic acid,L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine,L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine,L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.These amino acids may be obtained commercially, for example from Sigma(Saint Louis, Mo.).

Vitamin ingredients which may be included in the media of the presentinvention include biotin, choline chloride, D-Ca⁺⁺-pantothenate, folicacid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine andvitamin B₁₂. These vitamins may be obtained commercially, for examplefrom Sigma (Saint Louis, Mo.).

Inorganic salt ingredients which may be used in the media of the presentinvention include one or more calcium salts (e.g., CaCl₂), Fe(NO₃)₃,KCl, one or more magnesium salts (e.g., MgCl₂ and/or MgSO₄), one or moremanganese salts (e.g., MnCl₂), NaCl, NaHCO₃, Na₂HPO₄, and ions of thetrace elements selenium, vanadium and zinc. These trace elements may beprovided in a variety of forms, preferably in the form of salts such asNa₂SeO₃, NH₄VO₃ and ZnSO₄. These inorganic salts and trace elements maybe obtained commercially, for example from Sigma (Saint Louis, Mo.).

To this basal medium, one or more polyanionic or polycationic compoundsare added to formulate the complete culture media of the presentinvention; these compounds prevent the cells from clumping and promotegrowth of the cells in suspension. Thus, the complete media of theinvention are capable of supporting the suspension cultivation of amammalian cells in vitro. In the present media, the polyanionic compoundis preferably a polysulfonated or polysulfated compound, more preferablyheparin, dextran sulfate, heparan sulfate, dermatan sulfate, chondroitinsulfate, pentosan polysulfate, a proteoglycan or the like, which may beobtained from a number of commercial sources (such as Sigma; St. Louis,Mo.; Life Technologies, Inc.; Rockville, Md.). Particularly preferredfor use in the present culture media is dextran sulfate. Dextran sulfatemay be added to freshly formulated basal medium, or it may be formulatedas described in detail in Example 1 in a solution of basal medium(prepared as described above). This solution of dextran sulfate may alsobe prepared as a 1×-1000× formulation, most preferably as a 1×, 10×,100×, 500× or 1000× formulation, which is then diluted appropriatelyinto culture medium to provide a 1× final formulation in the completemedia of the present invention as described in detail in Example 1.

Dextran sulfate may be obtained commercially, for example from Sigma(Saint Louis, Mo.), and is preferably of an average molecular weight ofabout 5,000 to about 500,000 daltons, about 5,000 to about 250,000daltons, about 5,000 to about 100,000 daltons, about 5,000 to about50,000 daltons, about 5,000 to about 25,000 daltons, or about 5,000 toabout 10,000 daltons. Most preferably the dextran sulfate used in thepresent culture media is of a molecular weight of about 5,000 daltons.

Polyanionic and polycationic compounds can be used in accordance withthe invention for any cell, particularly epithelial cells or cell linesand fibroblast cells or cell lines, to prevent aggregation of clumpingof cells cultivated in suspension. Such cells include epithelial andfibroblast cells and cell lines. Such epithelial and fibroblast cellsare particularly those cells and cell lines described above, andpreferably, CHO cells, PER-C6 cells, and 293 cells).

To formulate the medium of the present invention, a polyanionic orpolycationic compound (and particularly, dextran sulfate) is added tothe above-described basal medium in an amount effective to preventclumping, or in an amount to effectively provide a suspension culture.e.g., at a concentration of about 0-500 mg/liter, about 1-250 mg/liter,about 5-200 mg/liter, about 10-150 mg/liter or about 50-125 mg/liter,and most preferably at a concentration of about 100 mg/liter. Similarconcentrations may be used for other polyanionic and polycationiccompounds, such as those described above, for formulating the completemedia of the invention.

The specific combinations of the above ingredients, their concentrationranges and preferred concentrations, in one example of the culture mediaof the invention are shown in Table 1. Although this specific exampleuses dextran sulfate, it is to be understood that any of theabove-described polyanionic or polycationic compounds may be used in thepresent media.

The above ingredients listed in Table 1, when admixed together insolution, form a complete culture medium of the present invention. Thesecomplete media are suitable for use in the culture of a variety ofmammalian cells, as described in more detail below. In particular, thecomplete media of the invention may be used to support cultivation ofmammalian cells in suspension, particularly those that ordinarily arecultivated in monolayers, as described below. However, the present mediaare also suitable for cultivating mammalian cells under standardmonolayer conditions.

TABLE 1 TYPICAL ANIMAL EPITHELIAL CELL CULTURE MEDIUM COMPONENTCONCENTRATIONS. Component A Preferred Most Preferred Ranges EmbodimentEmbodiment (mg/L) (mg/L) (mg/L) Component about: about: about: AminoAcids L-Alanine  0-100 0 0.00 L-Arginine 200-600 360 355.6 L-Asparagine 5-150 26 26.40 L-Aspartic Acid  10-350 75 75.00 L-Cysteine  15-150 5857.6 L-Glutamic Acid  5-150 30 29.40 L-Glutamine  300-1200 600 585.00Glycine  0-100 0 0.00 L-Histidine  25-200 42 42.2 L-Isoleucine  50-400190 190.00 L-Leucine 100-500 280 280.00 L-Lysine 100-500 200 204.0L-Methionine  25-250 115 115.00 L-Phenylalanine  15-200 70 70.00L-Proline  0-100 0 0.00 L-Serine  5-500 250 250.00 L-Threonine  15-40060 60.00 L-Tryptophan  5-100 20 20.00 L-Tyrosine  15-150 70 69.2L-Valine  50-500 200 190.00 Other Components Ethanolamine 0.5-5   3 3.2D-Glucose 2500-9000 4500 4500.00 HEPES 1000-7000 3000 2980.00 Insulin 5-25 10 10.00 Linoleic Acid 0.01-5.0  0.1 0.06 Lipoic Acid 0.2-15  22.00 Phenol Red 0.5-30  1 1.00 PLURONIC F68  0-750 300 300.00 Putrescine0.01-1   0.1 0.087 Sodium Pyruvate  10-500 110 110.00 Transferrin  3-1005 5.00 Vitamins Biotin 0.001-1    0.1 0.097 Choline Chloride  1-100 1414.00 D-Ca⁺⁺- 0.2-10  1 1.19 Pantothenate Folic Acid  1-100 5 5.00i-Inositol  2-200 18 18.00 Niacinamide 0.1-10  1 1.22 Pyridoxine 0.1-10 0.9 0.85 Riboflavin 0.02-5   0.2 0.22 Thiamine 0.1-10  1 1.00 VitaminB12 0.05-10   1 1.03 Dextran Sulfate,  50-250 100 100 MW 5000 InorganicSalts calcium salt  0-100 10 11.10 (e.g., CaCl₂) Fe(NO₃)₃ 0.25-1.5  0.80.810 KCl  10-500 275 276.30 MgCl₂  25-150 75 76.20 MgSO₄  5-150 2524.10 manganese salt 0.00001-0.0005  0.0001 0.0001 (e.g., MnCl₂) NaCl3000-9000 4400 4410.00 NaHCO₃  100-4000 2400 2400.00 Na₂HPO₄  10-750 125125.00 selenium salt 0.0000005-0.00002  0.000005 0.0000067 (e.g.,Na₂SeO₃) vanadium salt 0.00005-0.002  0.0006 0.0006 (e.g., NH₄VO₃) zincsalt (e.g., 0.001-0.15  0.1 0.0874 ZnSO₄)* *Concentrations of Fe(NO₃)₃and zinc salt(s) may be higher in protein-free complete media (seeabove).

For some applications, it may be preferable to further enrich thenutritional content of the complete media to support faster growth andenhanced production of biologicals by the cultured cells, and to providea more suitable environment for the culture of fastidious mammaliancells. To accomplish such enrichment, one or more supplements mayoptionally be added to the basal media or the complete media of theinvention. Supplements which may advantageously be added to the presentmedia include one or more cytokines (e.g., growth factors such as EGF,aFGF, bFGF, IGF-1, IGF-2, HB-EGF, KGF, HGF and the like), heparin (tostabilize heparin-binding growth factors such as the FGFs, HB-EGF, KGFand HGF) and one or more peptides derived from animals (e.g., HSA orBSA), yeasts (e.g., yeast extract, yeastolate or yeast extractultrafiltrate) or plants (e.g., rice or soy peptides). Cytokines, whichmay be natural or recombinant, are available commercially, for examplefrom Life Technologies, Inc. (Rockville, Md.) or R&D Systems, Inc.(Rochester, Minn.) and may be added to the basal media at concentrationsrecommended by the manufacturer for the particular cell type to becultured (typically a final concentration of about 0.00001-10 mg/liter).Heparin is available commercially, for example from Sigma (St. Louis,Mo.), and is preferably porcine mucosa heparin used at a finalconcentration in the media of about 1-500 U.S.P. units/liter. Animal,yeast and plant peptides may be obtained commercially (e.g., from Sigmafor animal peptides; from Difco, Norwell, Mass., for yeast peptides; andfrom Quest International, Norwich, N.Y., for plant peptides), or may bederived and formulated into the present culture media as described indetail in co-pending, commonly owned U.S. Application No. 60/028,197,filed Oct. 10, 1996, the disclosure of which is incorporated herein byreference in its entirety.

The basal and complete medium ingredients and optional supplements canbe dissolved in a liquid carrier or maintained in dry form. If dissolvedin a liquid carrier at the preferred concentrations shown in Table 1(i.e., a “1× formulation”), the pH of the medium should be adjusted toabout 7.0-7.6, preferably about 7.1-7.5, and most preferably about7.2-7.4. The osmolarity of the medium should also be adjusted to about260 to about 300 mOsm, preferably about 265 to about 280 mOsm, and mostpreferably about 265 to about 275 mOsm. The type of liquid carrier andthe method used to dissolve the ingredients into solution vary and canbe determined by one of ordinary skill in the art with no more thanroutine experimentation. Typically, the medium ingredients can be addedin any order.

Preferably, the solutions comprising individual ingredients are moreconcentrated than the concentration of the same ingredients in a 1×media formulation. The ingredients can be 10-fold more concentrated (10×formulation), 25-fold more concentrated (25× formulation), 50-fold moreconcentrated (50× concentration), or 100-fold more concentrated (100×formulation). More highly concentrated formulations can be made,provided that the ingredients remain soluble and stable. See U.S. Pat.No. 5,474,931, which is directed to methods of solubilizing culturemedia components at high concentrations.

If the individual medium ingredients are prepared as separateconcentrated solutions, an appropriate (sufficient) amount of eachconcentrate is combined with a diluent to produce a 1× mediumformulation. Typically, the diluent used is water but other solutionsincluding aqueous buffers, aqueous saline solution, or other aqueoussolutions may be used according to the invention.

The culture media of the present invention are typically sterilized toprevent unwanted contamination. Sterilization may be accomplished, forexample, by filtration through a low protein-binding membrane filter ofabout 0.22 μm or 0.45 μm pore size (available commercially, for example,from Millipore, Bedford, Mass.) after admixing the concentratedingredients to produce a sterile culture medium. Alternatively,concentrated subgroups of ingredients may be filter-sterilized andstored as sterile solutions. These sterile concentrates can then bemixed under aseptic conditions with a sterile diluent to produce aconcentrated 1× sterile medium formulation. Autoclaving or otherelevated temperature-based methods of sterilization are not favored,since many of the components of the present culture media are heatlabile and will be irreversibly degraded by temperatures such as thoseachieved during most heat sterilization methods.

The optimal concentration ranges for the basal medium ingredients arelisted in Table 1. These ingredients can be combined to form the basalmammalian cell culture medium which is then supplemented as describedabove with polyanionic or polycationic compounds (e.g., dextransulfate), and optionally with one or more supplements such as one ormore cytokines, heparin, and/or one or more animal, yeast or plantpeptides, to formulate the complete media of the present invention. Aswill be readily apparent to one of ordinary skill in the art, theconcentration of a given ingredient can be increased or decreased beyondthe range disclosed and the effect of the increased or decreasedconcentration can be determined using only routine experimentation. In apreferred embodiment, the concentrations of the ingredients of themedium of the present invention are the concentrations listed in the farright column of Table 1, supplemented with polyanionic or polycationiccompounds (e.g., dextran sulfate), and optionally with one or moresupplements such as one or more cytokines, heparin, and one or moreanimal, yeast or plant peptides, as described above.

As will be readily apparent to one of ordinary skill in the art, each ofthe components of the culture medium may react with one or more othercomponents in the solution. Thus, the present invention encompasses theformulations disclosed in Table 1, supplemented as described above, aswell as any reaction mixture (i.e., a culture medium or other reactionmixture) which forms after, or which is obtained by, combining theseingredients.

The optimization of the present media formulations was carried out usingapproaches described by Ham (Ham, R. G., Methods for Preparation ofMedia, Supplements and Substrata for Serum-Free Animal Culture, Alan R.Liss, Inc., New York, pp. 3-21 (1984)) and Waymouth (Waymouth, C.,Methods for Preparation of Media, Supplements and Substrata forSerum-Free Animal Culture, Alan R. Liss, Inc., New York, pp. 23-68(1984)). The optimal final concentrations for medium ingredients aretypically identified either by empirical studies, in single componenttitration studies, or by interpretation of historical and currentscientific literature. In single component titration studies, usinganimal cells, the concentration of a single medium component is variedwhile all other constituents and variables are kept constant and theeffect of the single component on viability, growth or continued healthof the animal cells is measured.

Formulation of the Replacement Culture Media

In the replacement media of the invention, any basal media may be used.Such basal media may contain one or more amino acids, one or morevitamins, one or more inorganic salts, one or more buffer salts, and oneor more lipids. In accordance with the invention, transferrin isreplaced with iron or an iron-containing compound and/or insulin isreplaced with zinc or a zinc containing compound. Preferably, ironchelate compounds are used in accordance with the invention

Fe²⁺ and/or Fe³⁺ chelate compounds which may be used include but are notlimited to compounds containing an Fe²⁺ and/or Fe³⁺ salt and a chelatorsuch as ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA),deferoxamine mesylate, dimercaptopropanol, diethylenetriaminepentaaceticacid (DPTA), and trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid(CDTA). For example, the iron chelate compound may be a ferric citratechelate or a ferrous sulfate chelate. Preferably, the iron chelatecompound used is ferrous sulphate-7H₂O.EDTA (FeSO₄.7H₂O.EDTA, e.g.,Sigma F0518, Sigma, St. Louis, Mo.). In the medium of the presentinvention, the concentration of Fe²⁺ and/or Fe³⁺ can be optimized usingonly routine experimentation. Typically, the concentration of Fe²⁺and/or Fe³⁺ in the 1× medium of the present invention can be about0.00028 to 0.011 g/L. Preferably, the concentration of iron is about0.0011 g/L.

Zn²⁺-containing compounds which may be used include but are not limitedto ZnCl, Zn(NO₃)₂, ZnBr, and ZnSO₄.7H₂O. Preferably, the Zn²⁺ compoundused is zinc sulfate.7H₂O (ZnSO₄.7H₂O). In the medium of the presentinvention, the concentration of Zn²⁺ can be optimized using only routineexperimentation. Typically, the concentration of Zn²⁺ in the 1× mediumof the present invention can be about 0.00007 to 0.00073 g/L.Preferably, the concentration of Zn²⁺ is about 0.000354 g/L.

The term “anticlumping agent” refers to a compound which reduces thedegree to which of cells in culture clump together. Preferably, theanticlumping agent is a polyanionic or polycationic compound. Thepolyanionic compound is preferably a polysulfonated or polysulfatedcompound, preferably dextran sulfate, pentosan polysulfate, heparin,heparan sulfate, dermatan sulfate, chondroitin sulfate, a proteoglycanor the like. More preferably, the anticlumping agent is dextran sulfateor pentosan polysulfate. Most preferably, the anticlumping agent isdextran sulfate, which preferably has a molecular weight of about 5,000daltons.

Anticlumping agents may be used to decrease clumping of cells grown insuspension culture, increase the level of recombinant proteinexpression, and/or virus production. The present invention provides aeukaryotic cell culture medium containing polyanionic or polycationiccompounds (preferably, dextran sulfate) in an amount sufficient toprevent cell clumping and/or increase the level of recombinant proteinexpression.

The inclusion of polyanionic or polycationic compounds (preferably,dextran sulfate) in the present media inhibits cell aggregation; thus,unlike traditional serum-free media in which suspension cells tend toaggregate or form clumps, the present media promote the cultivation ofsingle cells in suspension. The ability to cultivate cells under thesesuspension culture conditions provides for rapid subculturing andhigh-density culture, which are advantageous for applications in whichmammalian cells are used to produce a variety of products such as in thebiotechnology industry, as described below. Furthermore, since thepresent media are serum-free and low-protein or protein-free, the mediamay be used for rapid production and isolation of biologicals (e.g.,viruses, recombinant polypeptides, etc.), and in assays measuring thebinding and/or activity of a variety of ligands such as proteins,hormones, synthetic organic or inorganic drugs, etc., on mammalian cellsin vitro.

Dextran sulfate and pentosan polysulfate may be obtained commercially,for example from Sigma (St. Louis, Mo.). Dextran sulfate is preferablyof an average molecular weight of about 5,000 to about 500,000 daltons,about 5,000 to about 250,000 daltons, about 5,000 to about 100,000daltons, about 5,000 to about 50,000 daltons, about 5,000 to about25,000 daltons, or about 5,000 to about 10,000 daltons. Most preferablythe dextran sulfate used in the present culture media is of a molecularweight of about 5,000 daltons.

The pH of the 1× medium of the present invention should preferably bebetween about 6.9 to about 7.3. The osmolarity of the 1× medium of thepresent invention should preferably be between about 270 to about 350mOsm. If desired, the osmolarity can be increased by adding a suitablesalt such as NaCl. If the preferred concentrations of ingredients areused (Table 2), the osmolarity should not have to be adjusted.

Medium ingredients can be dissolved in a liquid carrier or maintained indry form. The type of liquid carrier and the method used to dissolve theingredients into solution vary and can be determined by one of ordinaryskill in the art with no more than routine experimentation.

Preferably, the solutions comprising ingredients are more concentratedthan the concentration of the same ingredients in a 1× mediaformulation. The ingredients can be 10 fold more concentrated (10×formulation), 25 fold more concentrated (25× formulation), 50 fold moreconcentrated (50× concentration), or 100 fold more concentrated (100×formulation). More highly concentrated formulations can be made,provided that the ingredients remain soluble and stable. See U.S. Pat.No. 5,474,931.

If the media ingredients are prepared as separate concentratedsolutions, an appropriate (sufficient) amount of each concentrate iscombined with a diluent to produce a 1× medium formulation. Typically,the diluent used is water, but other solutions including aqueousbuffers, aqueous saline solution, or other aqueous solutions may be usedaccording to the invention.

The culture medium of the present invention is typically sterilized toprevent unwanted contamination of the cell culture media. Sterilizationmay be accomplished, for example, by filtration after admixing theconcentrated ingredients to produce a sterile culture medium.Alternatively, concentrated subgroups of ingredients may befilter-sterilized and stored as sterile solutions. These sterileconcentrates can then be mixed with a sterile diluent to produce aconcentrated 1× sterile medium formulation.

The medium of the present invention facilitates the growth of mammaliancells, and particularly epithelial cells and cell lines, and fibroblastcells and cell lines, as described above, and particularly to highdensity, increases the level of expression of recombinant protein incultured cells, and/or increases virus production in cultured cells.Cellular metabolic energy is expended on both cell growth andrecombinant protein expression. Depending on the culture conditions andthe particular cell line, either cell growth or recombinant proteinexpression can be facilitated at the expense of the other activity.

To shift the distribution of metabolic energy from supporting cellgrowth to supporting protein expression, cells can be treated withsodium butyrate (De Gros, G. S. et al., Lymphokine Res. 4:221-227(1985)). About 10 μM to about 10-mM sodium butyrate can be used.Preferably, about 100 μM to 1.0 mM is used. Cells can be grown to thedesired density prior to the addition of sodium butyrate. After sodiumbutyrate has been added, cell growth slows and recombinant proteinexpression increases. Although treatment with sodium butyrate decreasesthe rate of cell growth, this decrease in growth rate is outweighed bythe increase in recombinant protein production. Moreover, because themedium of the present invention is protein-free, purification ofrecombinant protein can be performed more quickly, more easily and lessexpensively than purification can be done from cells that were grown inmedia containing serum or protein. See also U.S. Pat. No. 5,393,558.

Dihydrofolate reductase (DHFR) catalyzes the conversion of folate totetrahydrofolate, which is required for purine, amino acid, andnucleoside biosynthesis. The folic acid analogue methotrexate binds andinhibits DHFR, causing cell death. DHFR deficient cells (DHFR) whichhave been transfected with a gene of interest and a methotrexateresistance gene can be treated with methotrexate to amplify recombinantcells (Bebbington, C. R. et al., The Use of Vectors Based on GeneAmplification for the Expression of Cloned Genes n Mammalian Cells, In:DNA Cloning, Vol. III, Glover, D., ed., Academic Press (1987), pp.163-188). Surviving populations of cells exposed to sequentiallyincreasing concentrations of methotrexate contain increased levels ofDHFR that result from gene amplification.

About 50 nM to about 2 μM methotrexate can be used. Preferably, about100 nM to about 500 nM methotrexate is used. Although treatment withmethotrexate may decrease overall cell density, this decrease in celldensity is outweighed by the increase in recombinant protein production.Again, because the medium of the present invention is protein-free,purification of recombinant protein can be performed more quickly, moreeasily and less expensively than purification can be done from cellsthat were grown in media containing serum or protein.

The concentration ranges within which ingredients of the 1× medium arebelieved to support growth (and particularly the high-density growth),increase the level of expression of recombinant protein in culturedcells, and/or increases virus production in cultured, are listed in thesecond column of Table 2. These ingredients can be combined to form the1× eukaryotic cell culture medium of the present invention. As will beapparent to one of ordinary skill in the art, the concentration of agiven ingredient can be increased or decreased beyond the rangedisclosed and the effect of the increased or decreased concentration canbe determined using only routine experimentation. The concentration ofeach ingredient in a preferred embodiment of the medium of the presentinvention is listed in the third column of Table 2. The concentration ofeach ingredient in a particularly preferred embodiment is shown in thefourth column of Table 2.

The 1× medium of the present invention can be made using a mediumconcentrate technology. See U.S. Pat. No. 5,474,931. Ingredients can bestored in solution. Preferably, ingredients are grouped in concentratedsolutions and stored. For example, Table 2 shows suitable groups ofingredients. Stock solutions of the grouped ingredients can be made asconcentrated stocks. For example, it is possible to make 10× to 100×chemical stock solutions, which can be stored as liquids or frozen inthe appropriate aliquot sizes for later use.

Stock solutions offer a number of advantages. For example, a higherfinal concentration of a given ingredient can be used in the 1× medium.In addition, some ingredients are more stable when stored in aconcentrated stock solution. Moreover, less storage volume is requiredfor a concentrated stock solution than is required for a 1× medium. SeeU.S. Pat. No. 5,474,931.

In a preferred embodiment, 27.63× concentrated stock solutions of thegroups of ingredients in Table 2 are prepared as followed. To prepare a26.63× concentrated stock solution of the Acid Soluble I group ofingredients, each of the ingredients in the Acid Soluble I group ofingredients in Table 2 is added to approximately 19.33 mL of distilledwater. The ingredients in the Acid Soluble I group can be added in anyorder. The pH of the solution is reduced to 0.80 with 5N HCl(approximately 7.4 mL) and the solution is mixed until all of theingredients are dissolved. The final volume of the solution is 36.192mL.

To prepare a 27.63× concentrated stock solution of the Acid Soluble IIgroup of ingredients, sodium phosphate is first added to approximately39.33 mL of distilled water and the solution is mixed until the sodiumphosphate is completely dissolved. The pH is adjusted to 1.00 using 5NHCl (approximately 3.5 mL). The rest of the ingredients in the AcidSoluble II group in Table 2 are added (the order of addition of the restof these ingredients is not critical). A concentrated stock solution ofthe trace elements can be used. The Acid Soluble II solution is mixeduntil all of the ingredients are dissolved. The pH final should be 1.00.If necessary, the pH should be adjusted to 1.00. The final volume of thesolution is 36.192 mL.

To prepare a 27.63× concentrated stock solution of the Salts I solution,MgCl₂ and ascorbic acid Mg salt phosphate are added to approximately35.83 mL of distilled water. The solution is mixed until the ingredientsare dissolved. The pH is lowered to 5.5 using 5N HCl (approximately 6.0mL). D-Ca-pantothenate, calcium nitrate, and KCl are added and mixed.After mixing, the pH should be 5.50. If necessary, the pH should beadjusted to 5.50. The final volume of the solution is 36.192 mL.

To prepare a 27.63× concentrated stock solution of the Salts IIsolution, pluronic F-68 is added to approximately 28.23 mL of distilledwater, followed by sodium phosphate. After mixing, the pH is reduced to7.00 using 5N HCl (approximately 0.06 mL). Glucose is added, followed bythe rest of the ingredients in the Salts II group of ingredients inTable 2, except for folic acid and riboflavin (the order of addition ofthe rest of these ingredients, except for folic acid and riboflavin, isnot critical). Preferably, a 10,000× stock solution of sodium selenitein distilled water is used. The density of β-mercaptoethanol ispreferably 1.114 g/mL and the density of monothioglycerol is preferably1.250 g/mL.

In a separate container of 2 mL of distilled water protected from light,folic acid and riboflavin are added. The pH is adjusted to 11.5 using 5NHCl (approximately 7 μL) and the solution is mixed until the folic acidand riboflavin are dissolved. This solution of folic acid and riboflavinis then added to the Salts II ingredient solution. The final volume ofthe Salts II solution is 36.193 mL and the final pH should be 7.00. Ifnecessary, the pH can be adjusted to 7.00.

For the Salts II solution, the pluronic F-68 can be in liquid form or inpowder form. For the Acid Soluble I, Acid Soluble II, Salts I or Salts Hsolutions, unless other wise noted above, components can be added tosolution singly or in combination.

To prepare the 1× medium of the present invention, the followingprocedure is preferably used. To 840.000 mL of distilled water (pH 5.61)is added 36.192 mL of a 27.63× concentrate of the Acid Soluble I groupof ingredients (pH 1.77), followed by 36.192 mL of a 27.63× concentrateof the Acid Soluble II group of ingredients (pH 1.71), followed by about9.800 mL of 5N NaOH (pH 6.8), followed by 36.192 mL of a 26.63×concentrate of the Salts I group of ingredients (pH 6.8), followed by36.192 mL of a 26.63× concentrate of the Salts II group of ingredients(pH 6.85), followed by 1.810 mL of ferrous sulfate chelate (pH 6.85),followed by 2.22 g of NaHCO₃ (pH 7.16), followed by about 0.400 mL of 5NHCl (pH 7.00), followed by 0.427 g of NaCl. The final pH of the solutionshould be 7.00 and the final volume should be 1000 mL. The osmolarityrange of the solution should be between about 320 to 330 mOsm. 40 mL ofa 200 mM glutamine solution (100×) is added to the 1× medium at the timeof use.

The iron chelate compound is preferably added to the 1× medium prior tofilter sterilization.

Dextran sulfate can be added to the 1× medium to a final concentrationof about 1 μg/ml to about 1 mg/ml. Preferably, the final concentrationof dextran sulfate is about 10 to about 25 Dextran sulfate can be addedbefore filter sterilization of the 1× medium. Alternatively,presterilized dextran sulfate can be added to sterile 1× medium. Ifdextran sulfate is to be included in a concentrated stock solution, itcan be included in the Salts II group of ingredients (see Table 2). Theconcentration of other anticlumping agents can be determined using onlyroutine experimentation.

As will be apparent to one of ordinary skill in the art, the ingredientsmay react in solution. Thus, the present invention encompasses theformulations disclosed in Table 2 as well as any reaction mixture whichforms after the ingredients in Table 2 are combined.

TABLE 2 The 1X Replacement Medium Formulation PREFERRED PARTICULARLYCONCENTRATION EMBODIMENT PREFERRED RANGE (G/L) EMBODIMENT INGREDIENT(G/L) ABOUT (G/L) Acid Soluble I L-arginine 0.1000-0.7200 0.4 0.36192L-asparagine•H₂O 0.1000-1.8000 0.9 0.90480 L-aspartic acid 0.0100-0.36000.2 0.18096 L-glutamic acid 0.1000-0.6000 0.3 0.27144 L-histidine0.0600-0.3600 0.2 0.18096 hydroxy-L-proline 0.0040-0.3600 0.2 0.18096L-isoleucine 0.1000-0.7200 0.4 0.36192 L-leucine 0.1000-1.1000 0.50.54288 L-lysine•HCl 0.2000-1.1000 0.5 0.54288 L-methionine0.0500-0.2400 0.1 0.12667 L-phenylalanine 0.0900-0.4200 0.2 0.21715L-proline 0.0500-1.1000 0.5 0.54288 L-serine 0.1000-1.1000 0.5 0.54288L-threonine 0.1000-0.7200 0.4 0.36192 L-tryptophan 0.0200-0.4200 0.20.20810 L-tyrosine 0.1000-0.3600 0.2 0.18096 L-valine 0.1000-0.7200 0.40.36192 L-cystine•2HCl 0.0200-0.2200 0.1 0.10496 Acid Soluble II Na₂HPO₄(anhydrous) 0.2000-2.5000 0.6 0.63336 pyridoxine•HCl 0.0010-0.0072 0.0040.00362 thiamine•HCl 0.0010-0.0072 0.004 0.00362 glutathione0.0006-0.0036 0.002 0.00181 zinc sulfate•7H₂O 0.0003-0.0032 0.0020.00156 cupric sulfate•5H₂O 0.000001-0.000009 0.000005 0.000004524cadmium chloride•5H₂O 0.000004-0.000040 0.00002 0.000020629 cobaltchloride•6H₂O 0.0000006-0.0000086 0.000004 0.000004343 stannouschloride•2H₂O 0.00000001-0.00000020 0.0000001 0.000000101 manganoussulfate•H₂O 0.00000001-0.00000030 0.0000002 0.000000152 nickelsulfate•6H₂O 0.00000005-00000024   0.0000001 0.000000118 sodiummetavanadate 0.0000003-0000012   0.0000006 0.000000561 ammoniummolybdate•4H₂O 0.00000300-0.0000110  0.000005 0.000005429 barium acetate0.00000065-0.00000240 0.000001 0.000001176 potassium bromide0.00000003-0.00000011 0.00000005 0.000000054 potassium iodide0.000000045-0.00000016  0.00000008 0.000000081 chromium sulfate0.000000165-0.00000060  0.0000003 0.000000299 sodium fluoride0.00000105-0.00000360 0.000002 0.000001810 silver nitrate0.000000045-0.00000016  0.00000008 0.000000081 rubidium chloride0.00000035-0.0000013  0.0000006 0.000000633 zirconyl chloride0.0000008-0.0000029 0.000001 0.000001448 aluminum chloride0.0000003-0.0000011 0.0000005 0.000000543 germanium dioxide0.000000135-0.00000049  0.0000002 0.000000244 titanium tetrachloride0.00000025-0.0000009  0.0000005 0.000000452 sodium metasilicate0.00005-0.00095 0.0005 0.000452400 Salts I MgCL₂ (anhydrous)0.0100-0.1400 0.07 0.06985 D-Calcium pantothenate 0.0020-0.0060 0.0040.00362 Calcium nitrate•4H₂O 0.01800-0.3600  0.09 0.09048 KCl0.3340-1.4500 0.7 0.72384 Ascorbic acid, 0.00199-0.040  0.02 0.01991 Mgsalt phosphate Salts II Pluronic F68, 5.0 mL-40.0 mL/L 18 mL/L 18.096mL/L 10% Solution (0.5-4.0 g/L) (2 g/L) (1.8096 g/L) Na₂HPO₄ (anhydrous)0.018-0.360 0.09 0.09048 D-glucose 1.000-12.60 6 6.33360 folic acid 0.002-0.0072 0.004 0.00362 riboflavin  0.0002-0.00072 0.0004 0.000362biotin 0.000575-0.00360  0.002 0.00181 choline chloride 0.0280-0.18100.09 0.09048 niacinamide  0.0003-0.00724 0.004 0.00362 i-inositol0.0260-0.127  0.06 0.06334 sodium pyruvate 0.070-0.400 0.2 0.19906vitamin B-12 0.0005-0.0018 0.0009 0.00090 β-mercaptoethanol0.00014-0.00282 0.001 0.00141 para-aminobenzoic acid  0.0010-0.003620.002 0.00181 β-glycerophosphate 0.090-1.800 0.9 0.90480 sodium selenite0.00000157-0.000032  0.00002 0.0000157 ethanolamine•HCl 0.0075-0.02800.01 0.01357 spermine 0.0009-0.0181 0.009 0.00905 putrescine•2HCl0.00012-0.00110 0.0005 0.000543 monothioglycerol 0.0100-0.0362 0.020.01810 Dried powder medium NaHCO₃ 1-4 2 2.22

The iron chelate compound is preferably added to the 1× medium prior tofilter sterilization.

If glutamine is to be used, it can be added to the 1× medium prior tofilter sterilization. For example, 40 ml of 200 mM L-glutamine can beadded per liter of 1× medium (the final concentration of glutamine is 8mM). If glutamine is not added, then preferably the concentrations ofeach of the above ingredients should be diluted 1.04-fold/L withdiluent, although dilution is not necessary. Alternatively, a sterile200 mM stock solution of L-glutamine can be added after the 1× mediumhas been filter sterilized.

The 1× medium does not need to be supplemented with glutamine if theglutamine synthase expression system (Celltech, Slough, UK) is used.Using this system, cells are engineered to express glutamine synthetase,which catalyzes the synthesis of glutamine from glutamate and ammonia.See U.S. Pat. No. 5,122,464.

For the 1× medium to be effective for culturing NS/O myeloma cells, alipid mixture supplement may need to be added to the 1× medium. Thelipid supplement formulation of Table 3 can be added to the 1× mediumprior to filter sterilization. Preferably, a 200-400× concentrated stocksolution of the lipid mixture supplement is used. In a preferredembodiment, the lipid mixture supplement is prepared as a 379×concentrated stock solution of which 2.64 ml/L is added to the 1×medium. To make the lipid supplement, ethanol is added to Pluronic F68with constant stirring at 60° C. The mixture is cooled to 37-40° C. andthe sterol is added. The mixture is overlaid with argon gas and is mixeduntil the sterol dissolves. After the sterol has dissolved, the rest ofthe ingredients in Table 3 are added. This lipid mixture supplement canbe stored for future use. The sterol can be cholesterol or a plantsterol, such as sitosterol or stigmasterol or other plant sterol knownto those of ordinary skill in the art.

TABLE 3 Lipid supplement (final concentrations in 1X Replacementmedium). PARTICULARLY CONCENTRATION PREFERRED PREFERRED RANGE EMBODIMENT(G/L) EMBODIMENT INGREDIENT (G/L) ABOUT (G/L) Pluronic F68  0.1-4.0 1.71.74 ethanol  0.09-2.0 0.9 0.87 mL cholesterol 0.0007-0.07 0.007 0.00696lipoic acid 0.00003-0.003 0.0003 0.000296 linoleic acid 0.00001-0.0010.0001 0.000122 α-tocopherol 0.00002-0.002 0.0002 0.000231 palmitic acid0.0002-0.02 0.002 0.00174 oleic acid 0.0002-0.02 0.002 0.00174dilinoleoyl 0.0002-0.02 0.002 0.00174 phosphatidylcholine stearic acid0.0002-0.02 0.002 0.00174 linolenic acid 0.0002-0.02 0.002 0.00174palmitoleic acid 0.0002-0.02 0.002 0.00174 myristic acid 0.0002-0.020.002 0.00174

Kits

The invention also provides kits for use in the cultivation of amammalian cell, and in particular a mammalian epithelial cell. Kitsaccording to the present invention comprise one or more containerscontaining one or more of the media formulations of the invention. Forexample, a kit of the invention may comprise one or more containerswherein a first container contains a complete serum-free, low-protein orprotein-free culture medium prepared as described above.

Additional kits of the invention may comprise one or more containerswherein a first container contains a basal culture medium prepared asdescribed above and a second container contains a polyanionic orpolycationic compound, preferably a polysulfonated or polysulfatedcompound, more preferably heparin, dextran sulfate, heparan sulfate,dermatan sulfate, chondroitin sulfate, pentosan polysulfate, aproteoglycan or the like, and most preferably dextran sulfate. Thecomplete media, basal media and/or dextran sulfate contained in thecontainers of these kits may be present as 1× ready-to-use formulations,or as more concentrated solutions (for example 2×, 5×, 10×, 20×, 25×,50×, 100×, 500×, 1000× or higher). Additional kits of the invention mayfurther comprise one or more additional containers containing one ormore supplements selected from the group consisting of one or morecytokines, heparin, one or more animal peptides, one or more yeastpeptides and one or more plant peptides. Preferred cytokines, heparin,animal peptides, yeast peptides and plant peptides for inclusion in thecontainers of the kits of the invention are as described above. The kitsof the invention may be used to produce one or more of the culture mediaof the invention for use in a variety of applications as describedbelow.

Compositions

The invention further provides compositions comprising the media of thepresent invention. Compositions according to this aspect of theinvention may consist of one or more of the present media and optionallyone or more additional components, such as one or more cells, one ormore tissues, one or more organs, one or more organisms, one or moreviruses, one or more proteins or peptides (such as one or more enzymes),one or more hormones, one or more nucleic acids, one or more enzymesubstrates, one or more cytokines, one or more extracellular matrixcomponents (including attachment factors), one or more antibodies, oneor more detectable labeling reagents (such as fluorophores, phosphors orradiolabels), and the like.

Particularly preferred compositions of the invention comprise one ormore of the present culture media and one or more mammalian cells.Mammalian cells and cell lines, as described above, can be used in thecompositions of the present invention. Mammalian cells particularlysuitable for use in formulating the present cell culture compositionscomprising the suspension medium of the present invention includeepithelial cells of human origin, which may be primary cells derivedfrom a tissue sample such as keratinocytes, cervical epithelial cells,bronchial epithelial cells, tracheal epithelial cells, kidney epithelialcells or retinal epithelial cells, or transformed cells or establishedcell lines (e.g., 293 human embryonic kidney cells, HeLa cervicalepithelial cells or derivatives thereof (e.g., HeLaS3), PER-C6 humanretinal cells and HCAT human keratinocytes), or derivatives thereof.Most preferable for use in the present compositions of the suspensionmedium of the present invention are 293 human embryonic kidney cells.The cells used in such preferred compositions of the invention may benormal cells, or may optionally be diseased or genetically altered.Other mammalian cells, such as CHO cells, COS cells, VERO cells, BHKcells (including BHK-21 cells) and derivatives thereof, are alsosuitable for use in formulating the present cell culture compositions.

Mammalian cells and cell lines, as described above, can be used in thepresent compositions. Most preferable for use in the presentcompositions of the replacement medium of the present invention are CHOcells. The cells used in such preferred compositions of the inventionmay be normal cells, or may optionally be diseased or geneticallyaltered. Other mammalian cells, such as 293 cells, COS cells, VEROcells, BHK cells (including BHK-21 cells) and derivatives thereof, arealso suitable for use in formulating the present cell culturecompositions.

Epithelial tissues, organs and organ systems derived from animals orconstructed in vitro or in vivo using methods routine in the art maysimilarly be used to formulate the suspension and replacementcompositions of the present invention.

The compositions of the invention may be used in a variety of medical(including diagnostic and therapeutic), industrial, forensic andresearch applications requiring ready-to-use cultures, particularlysuspension cultures, of mammalian cells in serum-free, low-protein orprotein-free media. Non-limiting examples of uses of these compositionsinclude providing stock cell cultures; short- or long-term storage ofcells, tissues, organs, organisms and the like; providing vaccinationreagents; etc.

Use of Culture Media

The present cell culture media may be used to facilitate cultivation ofa variety of mammalian cells in suspension or in monolayer cultures. Inparticular, these media may be used to cultivate mammalian epithelialcells or cell lines, as described above, particularly human epithelialcells and cell lines and fibroblast cells and cell lines. The presentmedia advantageously facilitate the suspension cultivation of cellswhich are typically anchorage-dependent or grown in monolayer cultures,without the use of microcarriers such as latex or collagen beads(although cells may be cultivated on such microcarriers or beads in thepresent media). Methods for isolation, and suspension and monolayercultivation, of a variety of animal cells including mammalian epithelialcells are known in the art (see, e.g., Freshney, R. I., Culture ofAnimal Cells: A Manual of Basic Technique, New York: Alan R. Liss, Inc.(1983)) and are described in further detail below and in the Examples.While the present media are particularly useful for culturing mammaliancells in suspension, it is to be understood that the media may be usedin any standard cell culture protocol whether the cells are grown insuspension, in monolayers, in perfusion cultures (e.g., in hollow fibermicrotube perfusion systems), on semi-permeable supports (e.g., filtermembranes), in complex multicellular arrays or in any other method bywhich mammalian cells may be cultivated in vitro.

In a preferred embodiment, the replacement medium of the presentinvention is used to grow CHO cells in suspension culture. In anotherpreferred embodiment, the replacement medium of the present invention isused to grow hybridoma cells in suspension culture. In yet anotherpreferred embodiment, the replacement medium of the present inventioncan be used to culture NS/O myeloma cells in suspension culture. If NS/Omyeloma cells are cultured, the replacement 1× medium of the presentinvention can be supplemented with a lipid mixture supplement (see Table3).

The inclusion of a polyanionic or polycationic compound, such as dextransulfate, in the present media inhibits the aggregation of 293 cells, CHOcells, as well as the aggregation of other mammalian cells; thus, unliketraditional serum-free media in which suspension cells tend to aggregateor form clumps, the present media promote the cultivation of singlecells in suspension. The ability to cultivate cells under thesesuspension culture conditions provides for rapid subculturing andhigh-density culture, which are advantageous for applications in whichmammalian cells are used to produce a variety of products such as in thebiotechnology industry, as described below. Furthermore, since thepresent media are serum-free and low-protein or protein-free, the mediamay be used for rapid production and isolation of biologicals (e.g.,viruses, recombinant polypeptides, etc.), and in assays measuring thebinding and/or activity of a variety of ligands such as proteins,hormones, synthetic organic or inorganic drugs, etc., on mammalian cellsin vitro.

Cells which can be grown in the media of the present invention are thoseof animal origin, including but not limited to cells obtained frommammals. Mammalian cells particularly suitable for cultivation in thepresent media include epithelial cells and cell lines, which may beprimary cells derived from a tissue sample.

The media of the present invention may be used to culture a variety ofmammalian cells, including primary epithelial cells (e.g.,keratinocytes, cervical epithelial cells, bronchial epithelial cells,tracheal epithelial cells, kidney epithelial cells and retinalepithelial cells) and established cell lines (e.g., 293 embryonic kidneycells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK(NBL-1) cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Changcells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KBcells, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI 2650 cells,SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells,LLC-MK₂ cells, Clone M-3 cells, I-10 cells, RAG cells, TCMK-1 cells, Y-1cells, LLC-PK₁ cells, PK(15) cells, GH₁ cells, GH₃ cells, L2 cells,LLC-RC 256 cells, MH₁C₁ cells, XC cells, MDOK cells, VSW cells, andTH-I, B1 cells, or derivatives thereof), fibroblast cells from anytissue or organ (including but not limited to heart, liver, kidney,colon, intesting, esophagus, stomach, neural tissue (brain, spinalcord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue(lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, andfibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells,IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempseycells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells,Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38cells, WI-26 cells, MiCl₁ cells, CHO cells, CV-1 cells, COS-1 cells,COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB/3T3 cells,F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells,NOR-10 cells, C₃H/IOTI/2 cells, HSDM₁C₃ cells, KLN205 cells, McCoycells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L)cells, L-MTK⁻ (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, C_(II) cells,and Jensen cells, or derivatives thereof).

Cells may be normal cells, or may optionally be abnormal (e.g., diseasedor genetically altered). Other mammalian cells, such as leukemic celllines such as K562 cells, MOLT-4 cells, M1 cells and the like, andderivatives thereof, are also suitable for cultivation in the presentmedia.

293 human embryonic kidney cells and HeLaS3 cells are particularlypreferred for growth in the suspension medium of the present invention.Chinese hamster ovary (CHO) cells, NS/O cells, and hybridoma cells areparticularly preferred for growth in the replacement medium of thepresent invention. Especially preferred are CHO cells.

Cell lines and hybridoma lines are well known to those of ordinary skillin the art. See, for example, the ATCC Catalogue of Cell Lines andHybridomas, 7th Edition, 1992 (American Type Culture Collection,Rockville, Md., USA), and the ATCC Catalogue of Cell Lines andHybridomas, 8th Edition, 1996 (American Type Culture Collection,Rockville, Md., USA).

Epithelial tissues, organs and organ systems derived from animals orconstructed in vitro or in vivo using methods routine in the art maysimilarly be cultivated in the culture media of the present invention.

Isolation of Cells

Animal cells for culturing in the media of the present invention may beobtained commercially, for example from ATCC (Rockville, Md.), QuantumBiotechnologies (Montreal, Canada) or Invitrogen (San Diego, Calif.).Alternatively, cells may be isolated directly from samples of animaltissue obtained via biopsy, autopsy, donation or other surgical ormedical procedure.

Tissue should be handled using standard sterile technique and a laminarflow safety cabinet. In the use and processing of all human tissue, therecommendations of the U.S. Department of Health and HumanServices/Centers for Disease Control and Prevention should be followed(Biosafety in Microbiological and Biomedical Laboratories, Richmond, J.Y. et al., Eds., U.S. Government Printing Office, Washington, D.C. 3rdEdition (1993)). The tissue should be cut into small pieces (e.g.,0.5×0.5 cm) using sterile surgical instruments. The small pieces shouldbe washed twice with sterile saline solution supplemented withantibiotics as above, and then may be optionally treated with anenzymatic solution (e.g., collagenase or trypsin solutions, eachavailable commercially, for example, from Life Technologies, Inc.,Rockville, Md.) to promote dissociation of cells from the tissue matrix.

The mixture of dissociated cells and matrix molecules are washed twicewith a suitable physiological saline or tissue culture medium (e.g.,Dulbecco's Phosphate Buffered Saline without calcium and magnesium).Between washes, the cells are centrifuged (e.g., at 200×g) and thenresuspended in serum-free tissue culture medium. Aliquots are countedusing an electronic cell counter (such as a Coulter Counter).Alternatively, the cells can be counted manually using a hemacytometer.

Cultivation of Cells

The isolated cells and cell lines can be cultivated according to theexperimental conditions determined by the investigator. The examplesbelow demonstrate at least one functional set of culture conditionsuseful for cultivation of certain mammalian cells, particularly undersuspension conditions. It is to be understood, however, that the optimalplating and culture conditions for a given animal cell type can bedetermined by one of ordinary skill in the art using only routineexperimentation. For routine monolayer culture conditions, using themedia of the present invention, cells can be plated onto the surface ofculture vessels without attachment factors. Alternatively, the vesselscan be precoated with natural, recombinant or synthetic attachmentfactors or peptide fragments (e.g., collagen, fibronectin, vitronectin,laminin and the like, or natural or synthetic fragments thereof), whichare available commercially for example from Life Technologies, Inc.(Rockville, Md.), R&D Systems, Inc. (Rochester, Minn.), Genzyme(Cambridge, Mass.) and Sigma (St. Louis, Mo.). Isolated cells can alsobe seeded into or onto a natural or synthetic three-dimensional supportmatrix such as a preformed collagen gel or a synthetic biopolymericmaterial. For suspension cultivation, cells are typically suspended inthe present culture media and introduced into a culture vessel thatfacilitates cultivation of the cells in suspension, such as a spinnerflask, perfusion apparatus, or bioreactor (see Freshney, R. I., Cultureof Animal Cells: A Manual of Basic Technique, New York: Alan R. Liss,Inc., pp. 123-125 (1983)). Ideally, agitation of the media and thesuspended cells will be minimized to avoid denaturation of mediacomponents and shearing of the cells during cultivation.

The cell seeding densities for each experimental condition can beoptimized for the specific culture conditions being used. For routinemonolayer culture in plastic culture vessels, an initial seeding densityof 1-5×10⁵ cells/cm² is preferable, while for suspension cultivation ahigher seeding density (e.g., 5-20×10⁵ cells/cm²) may be used.

Mammalian cells are typically cultivated in a cell incubator at about37° C. The incubator atmosphere should be humidified and should containabout 3-10% carbon dioxide in air, more preferably about 8-10% carbondioxide in air and most preferably about 8% carbon dioxide in air,although cultivation of certain cell lines may require as much as 20%carbon dioxide in air for optimal results. Culture medium pH should bein the range of about 7.1-7.6, preferably about 7.1-7.4, and mostpreferably about 7.1-7.3.

Cells in closed or batch culture should undergo complete medium exchange(i.e., replacing spent media with fresh media) when the cells reach adensity of about 1.5-2.0×10⁶ cells/ml. Cells in perfusion culture (e.g.,in bioreactors or fermenters) will receive fresh media on a continuouslyrecirculating basis.

Virus Production

In addition to cultivation of mammalian cells in suspension or inmonolayer cultures, the present media may be used in methods forproducing viruses from mammalian cells. Such methods according to thisaspect of the invention comprise (a) obtaining a mammalian cell to beinfected with a virus; (b) contacting the cell with a virus underconditions suitable to promote the infection of the cell by the virus;and (c) cultivating the cell in the culture media of the invention underconditions suitable to promote the production of virus by the cell.According to the invention, the cell may be contacted with the viruseither prior to, during or following cultivation of the cell in theculture media of the invention; optimal methods for infecting amammalian cell with a virus are well-known in the art and will befamiliar to one of ordinary skill. Virus-infected mammalian cellscultivated in suspension in the media of the invention may be expectedto produce higher virus titers (e.g., 2-, 3-, 5-, 10-, 20-, 25-, 50-,100-, 250-, 500-, or 1000-fold higher titers) than those cells notcultivated in suspension in the media of the invention. These methodsmay be used to produce a variety of mammalian viruses and viral vectors,including but not limited to adenoviruses, adeno-associated viruses,retroviruses and the like, and are most preferably used to produceadenoviruses or adeno-associated viruses. Following cultivation of theinfected cells in the present media, the used culture media comprisingviruses, viral vectors, viral particles or components thereof (proteinsand/or nucleic acids (DNA and/or RNA)) may be used for a variety ofpurposes, including vaccine production, production of viral vectors foruse in cell transfection or gene therapy, infection of animals or cellcultures, study of viral proteins and/or nucleic acids and the like.Alternatively, viruses, viral vectors, viral particles or componentsthereof may optionally be isolated from the used culture mediumaccording to techniques for protein and/or nucleic acid isolation thatwill be familiar to one of ordinary skill in the art.

Recombinant Protein Production

The present culture media may also be used in methods for the productionof recombinant proteins from mammalian cells, particularly frommammalian cells grown in suspension. Cell lines commonly used forrecombinant protein production (e.g., CHO cells) typically produceproteins that are abnormally glycosylated (Lao, M.-S., et al.,Cytotechnol. 22:43-52 (1996); Graner, M. J., et al., Biotechnol.13:692-698 (1993); Graner, M. J., and Goochee, C. F., Biotechnol. Prog.9:366-373 (1993)). However, the relatively low β-galactosidase andsialidase activities in 293 cells at neutral pH, such as those providedby the present methods, may facilitate the production of recombinantproteins that more closely resemble their natural counterparts (Graner,M. J., and Goochee, C. F., Biotechnol. Bioeng. 43:423-428 (1994)).Furthermore, since the present culture media provide for rapid,high-density suspension cultivation of mammalian cells, the presentmethods facilitate the rapid production of recombinant proteins athigher concentrations than has been possible heretofore.

Methods of producing a polypeptide according to the invention comprise(a) obtaining a mammalian cell that has been genetically engineered toproduce a polypeptide; and (b) cultivating the mammalian cell in theculture media of the present invention under conditions favoringexpression of the polypeptide by the mammalian cell. Optimal methods forgenetically engineering a mammalian cell to express a polypeptide ofinterest are well-known in the art and will therefore be familiar to oneof ordinary skill. Cells may be genetically engineered prior tocultivation in the media of the invention, or they may be transfectedwith one or more exogenous nucleic acid molecules after being placedinto culture in the media. According to the invention, geneticallyengineered cells may be cultivated in the present culture media eitheras monolayer cultures, or more preferably as suspension culturesaccording to the methods described above. Following cultivation of thecells, the polypeptide of interest may optionally be purified from thecells and/or the used culture medium according to techniques of proteinisolation that will be familiar to one of ordinary skill in the art.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLES Materials and Methods

In each of the following examples, the following materials and methodswere generally used.

Unless otherwise indicated, all media and reagents were obtained fromLife Technologies, Inc. (Rockville, Md.).

Suspension Media Examples

Adenovirus type 5-transformed 293 human embryonic kidney epithelialcells were obtained from ATCC(CRL 1573), and were cultured as describedin Example 4 by incubation at 37° C. in a humidified atmosphereconsisting of 8% CO₂/92% air.

Replacement Medium Examples A. Media

CHO-S-SFM H (Life Technologies, Inc., Gaithersburg, Md.) is a lowprotein (<100 μg/ml) serum-free medium designed for growth andrecombinant protein expression by CHO cells in suspension culture.CHO-S-SFM-II contains both insulin and transferrin.

CHO III Prototype is an essentially protein-free formulation whichcontains no animal-derived proteins and is designed for growth andrecombinant protein expression in suspension culture. The terms “CHO IIIPrototype,” CHO III PFM,” and “CHO III” are synonymous.

CD CHO is a particularly preferred embodiment of the replacement mediumof the present invention (Table 1, the particularly preferred embodimentcolumn), to which a Fe²⁺ and/or Fe³⁺ chelate has been added, and is achemically defined formulation designed for suspension cultureapplications. Where applicable, glutamine is also added.

All three of the above media were formulated without hypoxanthine andthymidine for DHFR amplified rCHO cultures.

B. CHO Cells

Wild-type CHO DG44 cells were obtained from Dr. Lawrence Chasin(Columbia University) and were adapted to suspension culture inCHO-S-SFM II supplemented with hypoxanthine and thymidine. Cells weremaintained in CHO-S-SFM II+HT Supplement or CHO III Prototype (LifeTechnologies, Inc.).

To establish a recombinant bovine growth hormone (rbGH) CHO cell line,wild-type CHO-K1 cells were transfected with two plasmids: pRSVneo,containing a neomycin resistance gene, and a bGH cassette using theprotocol supplied with LipofectAMINE™ Reagent (Life Technologies).Selection was conducted in the presence of the neomycin analogue, G418,at a concentration of 1.2 mg/ml. Stock cultures of rCHO cells weremaintained in either CHO-S-SFM II or CHO III Prototype supplemented with0.6 mg/ml G418 (all products from Life Technologies, Inc.).

To establish a recombinant β-galactosidase (rβ-Gal) CHO cell line, CHOcells deficient in dihydrofolate reductase (DHFR⁻) were obtained fromATCC (CRL-9096, Rockville, Md.) and transfected with two plasmids:pSV2dhfr (ATCC 37146), containing a gene for methotrexate (MTX)resistance, and pCMVβgal, which contains the lacZ cDNA. The transfectionwas conducted using LipofectAMINE™ Reagent, and selection wasaccomplished with 1.2 μM methotrexate (Sigma Chemical Co., St Louis,Mo.). Stock cultures were maintained in CHO-S-SFM II or CHO IIIPrototype supplemented with 0.3 μM MTX.

C. Assays

1. Recombinant bGH Quantitation by ELISA

rbGH production was quantitated using the Non-Isotopic ImmunoassaySystem for bGH Transfection Protein™ (Life Technologies Inc.,Gaithersburg, Md.) following manufacturer's instructions.

2. rβ-Gal Assay

rβ-Galactosidase (rβ-gal or rβeta-Gal) was measured in cell lysatesusing a modification of the method described by Hall et al., J. Mol.Appl. Gen. 2:101-109 (1983) and Miller, Experiments in MolecularGenetics, Cold Spring Harbor Laboratory (1972). Briefly, a 2.0 ml sampleof cell suspension was collected from each sample and subjected to twofreeze-thaw cycles, centrifuged at 100×g for 4 minutes, and thesupernatant was saved for rβ-Galactosidase quantitation. 100 μl of eachsupernatant sample was added to 1.0 ml Z buffer (0.06 M Na₂HPO₄, 0.04MNaH₂PO₄, 0.01M KCl, 0.01M MgSO₄.7H₂O, 0.05M β-mercaptoethanol, pH 7.0),followed by 200 μl of o-nitrophenyl-β-D-galactoside (ONPG, 4.0 mg/ml indH₂0) and incubated at 37° C. for 120 minutes. The reaction was haltedby addition of 500 μl stop buffer (1M Na₂CO₃) and absorbance at 420 nmwas read against a blank of the appropriate medium. Activity wascalculated using the following formulae:

$\begin{matrix}{{1.\mspace{14mu} \frac{1000 \times A_{120}}{120\mspace{14mu} \min \times 0.1\mspace{14mu} {ml}}} = {{{Units}/r}\; \beta \text{-}{galactosidase}}} \\{{2.\mspace{14mu} {Units}\mspace{14mu} r\; \beta \text{-}{{galactosidase}/\#}\mspace{14mu} {of}\mspace{14mu} {cells}} = {{Activity}/{cell}}}\end{matrix}$

rβ-Gal was detected in cells using the staining method of Sanes et al.,EMBO J. 5:3133-3142 (1986).

3. Amino Acid, Ammonia, Glucose and Lactate Measurements

Amino acid and ammonia levels in culture supernatants were measured byHPLC using the Waters AccQ-Tag method (Millipore Corp., Milford, Pa.).Glucose and Lactate concentrations were determined with the YSI SelectBiochemistry Analyzer (Model 2700, YSI, Yellow Springs, Ohio).

D. CHO Cell Culture

Unless indicated otherwise below, CHO cell culture conditions were asfollows for the following Examples. 25-35 mL of CHO cells were culturedin 125 mL shake flasks in humidified air containing 5-10% carbondioxide. The shake flasks were shaken on an orbital shaking platform at125-135 rpm. Temperature was maintained at 37° C. Cells were subculturedevery three to four days to a density of 2-3×10⁵ cells/mL.

Example 1 Formulation of Complete Suspension Medium

Formulation of Basal Cell Culture Medium.

To formulate the basal cell culture medium, the following were blendedas powders: L-arginine.HCl (430.00 mg/L; 355.6 mg/L L-arginine freebase), L-asparagine (anhydrous) (26.40 mg/L), L-aspartic acid (75.00mg/L), L-cysteine (57.6 mg/L), L-glutamic acid (29.40 mg/L), L-glutamine(585.00 mg/L), L-histidine (42.15 mg/L), L-isoleucine (190.00 mg/L),L-leucine (280.00 mg/L), L-lysine (204 mg/L), L-methionine (115.00mg/L), L-phenylalanine (70.00 mg/L), L-serine (250.00 mg/L), L-threonine(60.00 mg/L), L-tryptophan (20.00 mg/L), L-tyrosine (69.2 mg/L),L-valine (190.00 mg/L), biotin (0.097 mg/L), D-Ca⁺⁺-pantothenate (1.19mg/L), choline chloride (14.00 mg/L), folic acid (5.00 mg/L), i-inositol(18.00 mg/L), niacinamide (1.22 mg/L), pyridoxine.HCl (1.03 mg/L; 0.85mg/L pyridoxine free base), riboflavin (0.22 mg/L), thiamine (0.99mg/L), vitamin B₁₂ (1.03 mg/L), putrescine (0.087 mg/L), D-glucose(4500.00 mg/L), KCl (276.30 mg/L), NaCl (4410.00 mg/L), HEPES (2980.00mg/L), linoleic acid (0.06 mg/L), D,L-lipoic acid (2.00 mg/L), phenolred (1.00 mg/L), PLURONIC F68 (300.00 mg/L), sodium pyruvate (110.0mg/L), Na₂HPO₄ (125.00 mg/L), insulin (zinc human recombinant) (10.00mg/L), transferrin (human hobo-, heat-treated) (5.00 mg/L),ethanolamine.HCl (5 mg/L; 3.2 mg/L ethanolamine), Fe(NO₃)₃.9H₂O (0.810mg/L), MgCl₂ (76.20 mg/L), MgSO₄ (24.10 mg/L), CaCl₂ (11.10 mg/L),ZnSO₄.H₂O (0.0874 mg/L), Na₂SeO₃ (0.0000067 mg/L), MnCl₂ (0.0001 mg/L)and NH₄VO₃ (0.0006 mg/L).

NaHCO₃ (2400.00 mg/L) was added to the medium solution, and the pH ofthe solution was then adjusted with HCl to 7.2±0.05 and the volumeadjusted to the full desired volume with ddH₂O. The osmolality wasdetermined to be 265-275 mOsm.

To formulate the complete culture medium, 100 mg/L dextran sulfate(average molecular weight=5,000 daltons) were added to the basal medium,and the complete medium was filtered through a low protein-bindingfilter and used immediately or stored at 4° C. under diminished lightconditions until use.

Example 2 Formulation of Lower Protein and Protein-Free Culture Media

To produce a culture medium that was lower in protein, a basal mediumwas formulated as described in Example 1 except that transferrin wasomitted from the formulation. In place of transferrin, either 40 μMFeSO₄-EDTA (Sigma; St. Louis, Mo.) or 60 μM FeCl₃-sodium citrate (Sigma)were added to the basal media, and this lower protein medium was thenfiltered and stored as described in Example 1.

To formulate a culture medium that is completely free of protein, alower protein medium containing no transferrin is produced as describedabove, except that insulin is also omitted from the formulation and,instead, the final concentration of ZnSO₄ is increased to 0.354 mg/L asa substitute for insulin. Protein-free culture media are then filteredand stored as described in Example 1.

Example 3 Enrichment of Culture Medium

To provide a more enriched culture medium, the basal and/or completemedia described above were supplemented with additional components. Inone such enrichment, resulting in a culture medium that was low inanimal protein (or animal protein-free), a formulation of hydrolyzedrice peptides (Hy-Pep Rice Extract; Quest International, Norwich, N.Y.)was added to the complete media of Example 1 or Example 2 (for animalprotein-free media) at a concentration of about 100 mg/L (concentrationsof about 1-1000 mg/L were found to be acceptable as well). Enrichedculture media were then filter-sterilized and stored until use asdescribed in Example 1. In an alternative formulation providing anenriched culture medium that is free of animal protein, a formulation ofsoy peptides (Hy-Soy; Quest International) is added at similarconcentrations.

Other enriched culture media are prepared by adding one or morecytokines, such as growth factors at the following optimalconcentrations: EGF (about 0.00001-10 mg/L), aFGF or bFGF (about0.0001-10 mg/L), KGF (about 0.0001-10 mg/L) or HGF (about 0.00001-10mg/L). Other cytokines are also optionally added at concentrations thatare easily optimized by routine experimentation (e.g., dose-responsecurves). Cytokines are available commercially, for example, from LifeTechnologies, Inc. (Rockville, Md.).

Other enriched culture media are prepared by adding one or more animalpeptides, such as bovine serum albumin (BSA), human serum albumin (HSA)or casein. Animal peptides are available commercially, for example fromSigma (St. Louis, Mo.), and are added to basal media or complete mediaat concentration ranges of about 1-30,000 mg/L; optimal concentrationsfor a particular application are easily determined by routineexperimentation.

Other enriched culture media are prepared by adding one or more yeastpeptides, such as yeast extract, yeastolate or yeast extractultrafiltrate. Yeast extract and yeastolate are available commercially,for example from Difco (Norwell, Mass.), while yeast extractultrafiltrate is prepared as described in U.S. Application No.60/028,197, filed Oct. 10, 1996, the disclosure of which is incorporatedherein by reference in its entirety. Yeast peptides are added to basalor complete media at concentration ranges of about 1-30,000 mg/L;optimal concentrations for a particular application are easilydetermined by routine experimentation.

Following preparation, enriched culture media are filter sterilized andstored as described in Example 1 for complete medium.

Example 4 Use of Culture Media for Suspension Culture of EpithelialCells

To demonstrate the efficacy of the present media in suspension cultureof anchorage-dependent cells, 293 human embryonic kidney cellstransformed with adenovirus type 5 DNA were cultivated. Cultures of 293cells in serum-supplemented media were weaned from serum by passage 2-3times in OptiMEM medium (Life Technologies, Inc.; Rockville, Md.)supplemented with 2% normal horse serum in 165 cm² culture flasks. Afterthe second or third passage, when cells reached 50-75% confluence, theywere dislodged from the growth surfaces by gently rapping the flasksseveral times; trypsin or other proteolytic agents were not used, sincesuch agents often cause irreversible damage to cells cultivated inlow-serum or serum-free media. Cells were resuspended in the presentculture media and triturated until aggregates dispersed into a singlecell suspension, and cell concentration and viability was determined bytrypan blue exclusion counting on a hemacytometer according to routineprocedures.

Cells were then seeded into the present culture medium in Erlenmeyerflasks at a density of about 2.5-3.0×10⁵ cells/ml. To minimizeaggregation of the cells, the seed volume in the culture flasks was keptbelow about 20% of the total volume of the flask (e.g., for 125 mlflasks, total volume after dilution of cells was no more than about21-22 ml; for 250 ml flasks, no more than about 40-45 ml). To maintaincells in suspension, flasks were then placed on a rotary shaker platformin an incubator (37° C., 8% CO₂/92% air) and shaken at 125 rpm. Celldensities and viabilities were determined at least every other day, andcells were subcultured when the density approached about 7.5-10.0×10⁵cells/ml by diluting cultures with fresh culture medium to a density ofabout 2.5-3.0×10⁵ cells/ml. Subculturing was continued until aggregationof cells during cultivation appeared minimal.

Once cells were adapted to cultivation in suspension in the presentculture media, the cultures were scaled up into larger-volume spinnerflasks or bioreactors. Cells were concentrated from flask cultures bycentrifugation at about 400 g for five minutes, pellets were resuspendedby gentle trituration in the present media followed by vortex mixing for45 seconds, and cells seeded into spinner flasks or bioreactors in thepresent media at a density of about 2.5-3.0×10⁵ cells/ml. To minimizeshearing of cells while maintaining the cells in suspension, for spinnercultures the spinner speed was set to about 150 rpm while for bioreactorcultures the impeller speed was set to about 70 rpm.

In Erlenmeyer flask cultures of 293 cells, viable cell densities ofabout 2.5-3.0×10⁶ cells/ml were obtained (data not shown). As shown inFIG. 1, cell densities of up to 3.5-4.0×10⁶ cells/ml, with nearly 100%viability, were obtained in bioreactors and spinner cultures of 293cells in the complete media of the invention within 2-3 days afterinitiation of the cultures. Similar results were obtained withsuspension cultures of HeLaS3 cervical epithelial cells (not shown).Lower-protein culture media of the invention, in which transferrin hadbeen replaced by either FeSO₄-EDTA or FeCl₃-sodium citrate chelates,performed equivalently to complete media in supporting 293 cell growth(FIG. 2).

Together, these results demonstrate that the present culture mediapromote high-density suspension culture of anchorage-dependentepithelial cells and 293 cells in a serum-free, low-protein orprotein-free environment.

Example 5 Production of Viruses by Suspension Cultures of 293 Cells

To examine the utility of the present media in virus productionprotocols, suspension cultures of 293 cells were prepared as describedin Example 4 and infected with adenovirus. Infection of cells wasperformed directly in the suspension cultures by adding adenovirus at aMOI of about 5-50, and cultures were then maintained as in Example 4 forabout 48-96 hours. Adenovirus was then harvested with the culture mediumand titered on the permissive host cell line A549.

As shown in FIG. 3, adenovirus-infected 293 cells cultured in the mediaof the invention produced high titers of adenovirus-5 beginning atapproximately day 2 post-infection and continuing through day 4. Theseresults demonstrate that the present culture media facilitate the rapid,large-scale production of active viruses, such as adenovirus, insuspension cultures of 293 cells.

Example 6 Expression of Exogenous Genes by Suspension Cultures of 293Cells

The utility of the present media in facilitating the production ofrecombinant polypeptides was also examined. Line 293 cells were platedin RPMI-1640 containing 10% FBS (growth medium) in six-well cultureplates at 3×10⁵ cells/ml, and one day later were transfected with 1.5 μgof pCMV.SPORT-β-galactosidase and 0.5 μg of pSV2neo plasmid DNAs (LTI;Rockville, Md.) in the presence of 12 μl of LipofectAMINE (LTI) usingthe standard LipofectAMINE protocol. At 24 hours post-transfection,cells in each well were subcultured into growth medium in a 10 cm² plateat final dilutions of 1:50, 1:200 and 1:500, and at 48 hourspost-transfection the growth medium was replaced with selection medium(growth medium containing 500 μg/ml genetecin (LTI)). Medium was changedas necessary to remove nonviable cells, and resistant clones wereselected with cloning cylinders and transferred to 24-well plates foradaptation to suspension culture.

Clones expressing β-galactosidase were adapted to suspension culture inthe present culture media according to the procedures described indetail in Example 4. For all cultures, to maintain selective pressure onthe cells, genetecin was included in the present culture media atconcentrations of up to 50 μg/ml. Higher concentrations of geneticin arediscouraged, since they may be toxic in serum-free, low-protein mediasuch as those of the present invention.

As shown in FIG. 4, significant quantities of β-galactosidase wereproduced by transfected 293 cells cultured in the present media,beginning at day 1 post-transfection and continuing throughout the sixday course of the culture. These results demonstrate that the presentculture media facilitate stable transfection of epithelial cells withexogenous genes, and the rapid, large-scale production of recombinantpolypeptides by suspension cultures of 293 cells.

Example 7

rβ-gal CHO cells were planted at 3×10⁵ cells/ml in CHO-S-SFM II medium(without hypoxanthine and thymidine), CD CHO medium, or CHO IIIPrototype medium. Samples were saved at various time points fordetermination of cell density and β-galactosidase expression levels. Asshown in FIG. 5A, the highest cell density was obtained in CD CHOmedium, which is the medium of the present invention. At day 4 ofculturing, the highest level of β-galactosidase expression was observedin CHO III medium (FIG. 5B). By day 6 of culturing, whereas the level ofβ-galactosidase expression in cells grown in CHO III medium haddeclined, the level of β-galactosidase expression in cells grown in thereplacement medium of the present invention (CD CHO medium) hadincreased. Indeed, the level of β-galactosidase expression in cellsgrown in CD CHO medium continued to increase at day 7 of culturing.

Example 8

rβ-gal CHO cells were planted at 2×10⁵ cells/ml in either CHO-S-SFM IImedium (without hypoxanthine and thymidine), CD CHO medium, or CHO IIImedium. On day 3 post-planting, when the CD CHO cultures had reachedapproximately 1.2×10⁶ cells/ml, the CD CHO cultures were centrifuged andresuspended in fresh CD CHO or CHO III media. By day 8, the CD CHOculture that had been changed to CHO III medium had reached a lower peakcell density than the culture kept in CD CHO medium (FIG. 6A).

At day 7 of culturing, the level of rβ-galactosidase expression in cellsgrown in the replacement medium of the present invention (CD CHO medium)was comparable to the level in cells that were switched to CHO IIImedium (FIG. 6B).

Example 9

rβ-gal CHO cells were planted at 1.8×10⁵ cells/ml in CD CHO mediumsupplemented with increasing concentrations of methotrexate (MTX).Concentrations indicated are final concentrations. Samples were takendaily for determination of cell density and rβ-galactosidase expression.Although MTX concentration was inversely proportional to cell density(FIG. 7A), MTX concentration was proportional to rβ-galactosidasespecific activity (FIG. 7B). Thus, the medium of the present invention,when supplemented with methotrexate, can be used to grow recombinantDHFR amplified CHO cells which express high levels of recombinantprotein. Because the medium of the present invention is protein-free,the recombinant protein product can be easily purified.

Example 10

rbGH CHO cells were planted at 2×10⁵/ml in 125 ml shake flasks (35 mlvolume) in either CHO III medium or CD CHO medium. Daily samples weretaken for determination of viable cell density and rbGH levels. The CDCHO cultures reached a higher peak cell density (FIG. 8A) and expressedhigher levels of rbGH (FIG. 8B).

Example 11

rβ-gal CHO cells were planted at 3×10⁵/ml in 125 ml shake flasks (20-35ml volume) in either CHO-S-SFM II medium or CD CHO medium. Daily sampleswere taken for determination of viable cell density and rβ-gal levels.Total cell density continued to rise in CD CHO cultures (days 3 through8, FIG. 9A), while total cell density remained constant in CHO-S-SFM IIculture. rβ-gal levels continued to rise in both culture conditions, butCD CHO cultures expressed more total rβ-gal product (FIG. 9B). Thus,compared to a medium that contains insulin and transferrin, the mediumof the present invention supports increased cell growth and level ofprotein expression.

Example 12

rβ-gal CHO cells were planted at 3×10⁵/ml in 125 ml shake flasks (20-35ml volume) in CD CHO, CHO III PFM, or FMX-8 media. FMX-8 medium isdisclosed in Zang, M. et al., Bio/Technology 13:389-392 (1995).

As shown in FIG. 10, after seven days of culturing, cells grown in CHOIII PFM medium grew to a density about two-fold that of cells grown inFMX-8 medium. As shown in FIG. 10, cells grown in CD CHO medium grew toa density about three-fold that of cells grown in CD CHO medium andabout six-fold that of cells grown in FMX-8 medium.

As shown in FIG. 11, cells grown in CHO III PFM medium expressed rβ-galat a level about three-fold that of cells grown in FMX-8 medium. Asshown in FIG. 11, cells grown in CD CHO medium expressed rβ-gal at alevel about 1.6-fold that of cells grown in CD CHO medium and aboutfive-fold that of cells grown in FMX-8 medium. Thus, compared to theFMX-8 medium, the medium of the present invention supports increasedlevels of cell growth and protein expression.

Example 13

The CD CHO medium supports scaled-up cultures of mammalian cells aswell. rβ-gal CHO cells were planted at 1-3×10⁵/ml in 250 ml shake flasks(75 ml working volume) in CD CHO medium and cultured at pH 7.40, 50% airsaturation, 37° C., while shaking at 125-135 r.p.m. For bioreactorexperiments, rβ-gal cells were planted at 1-3×10⁵/ml in a 5 L stirredtank Celligen bioreactor (3.8 L working volume) in CD CHO medium andcultured at pH 7.40, 50% air saturation, 37° C., while stirring at 90r.p.m. The arrow indicates supplementation with 3 g/L glucose (finalconcentration) and 1 g/L glutamine (final concentration) at day nine ofculturing.

As shown in FIG. 12A, the growth kinetics of rCHO cells cultured in thebioreactor were similar to those observed in the shake flask. As shownin FIG. 12B, the level of rβ-gal expression was higher in cells culturedin the bioreactor. Supplementation with glucose and glutamine did notboost cell growth over the level reached on day nine. These resultsindicate that the CD CHO medium can be used successfully in scaled upcell culture.

Example 14

To determine the effect of the anticlumping agent dextran sulfate oncell growth and protein expression, recombinant cells were cultured inthe presence or absence of dextran sulfate (molecular weight of either5,000 or 500,000). rβ-gal CHO cells were planted at 3×10⁵/ml in 125 mlshake flasks (20-35 ml volume) in CD CHO medium. Dextran sulfate wasadded to the medium, at the time of cell planting, to a finalconcentration of 25 μg/mL. Results are shown in FIGS. 13 and 14. InFIGS. 13 and 14, “A” is dextran sulfate (m.w. 5,000) and “C” is dextransulfate (m.w. 500,000). CD CHO Control cells are cells to which dextransulfate was not added. As shown in FIG. 13, cells grown mediumcontaining dextran sulfate (m.w. 5,000) displayed increased cell growthand viability at days 5, 7, and 9. As shown in FIG. 13, cells grown indextran sulfate (m.w. 500,000) displayed increased cell growth andviability at days 5 and 7, but not at day 9. As shown in FIG. 14, cellsgrown in dextran sulfate (m.w. 5,000) displayed an increase in the levelof rβ-gal expression at days 5, 7, and 9. As shown in FIG. 14, cellsgrown in medium supplemented with dextran sulfate (m.w. 500,000) did notdisplay enhanced expression.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1-78. (canceled)
 79. A method of producing a polypeptide comprising (a)obtaining a mammalian cell that has been genetically engineered toproduce a polypeptide; and (b) cultivating said cell in a serum-free andprotein-free cell culture medium comprising an insulin substitute and atransferrin substitute, under conditions favoring expression of saidpolypeptide by said mammalian cell.
 80. The method of claim 79, whereinsaid mammalian cell is an epithelial cell.
 81. The method of claim 79,wherein said mammalian cell is a human cell.
 82. The method of claim 81,wherein said human cell is a 293 embryonic kidney epithelial cell.
 83. Apolypeptide produced according to the method of claim
 79. 84. Aeukaryotic cell culture medium comprising a Fe²⁺ chelate and a Zn²⁺salt, wherein said medium is capable of supporting the high-densitygrowth of mammalian cells in suspension culture and/or the expression ofrecombinant protein.
 85. (canceled)
 86. The eukaryotic cell culturemedium according to claim 84, wherein said mammalian cells are Chinesehamster ovary cells.
 87. The eukaryotic cell culture medium according toclaim 84, wherein said medium is a 1× medium formulation.
 88. Theeukaryotic cell culture medium according to claim 84, wherein saidmedium is a concentrated medium formulation.
 89. The eukaryotic cellculture medium according to claim 88, wherein said medium is a 10×medium formulation.
 90. The eukaryotic cell culture medium according toclaim 88, wherein said medium formulation is greater than 10×. 91-97.(canceled)
 98. The eukaryotic cell culture medium according to claim 84,further comprising one or more ingredients selected from the groupconsisting of L-arginine, L-asparagine. H₂O, L-aspartic acid, L-glutamicacid, L-histidine, hydroxy-L-proline, L-isoleucine, L-leucine,L-lysine.HCl, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cystine.2HCl,Na₂HPO₄, pyridoxine. HCl, thiamine. HCl, glutathione, cupricsulfate.7H₂O, cadmium chloride.5H₂O, cobalt chloride.2H₂O, stannouschloride. 2H₂O, manganous sulfate. H₂O, nickel sulfate. 6H₂O, sodiummetavanadate, ammonium molybdate. 4H₂O, barium acetate, potassiumbromide, potassium iodide, chromium sulfate, sodium fluoride, silvernitrate, rubidium chloride, zirconyl chloride, aluminum chloride,germanium dioxide, titanium tetrachloride, sodium metasilicate,magnesium chloride (anhydrous), D-calcium pantothenate, calcium nitrate.4H₂O, potassium chloride, ascorbic acid magnesium salt phosphate,pluronic F68 10% solution, Na₂HPO₄, D-glucose, folic acid, riboflavin,biotin, choline chloride, niacinamide, i-inositol, sodium pyruvate,vitamin B-12, β-mercaptoethanol, para-aminobenzoic acid,β-glycerophosphate, sodium selenite, ethanolamine.HCl, spermine,putrescine.2HCl, monothioglycerol, and sodium bicarbonate, wherein eachof said ingredients is present in an amount which supports thehigh-density growth of Chinese hamster ovary cells in suspension cultureand/or the expression of recombinant protein.
 99. A eukaryotic cellculture medium obtained by combining ferrous sulfate.EDTA andZnSO₄.7H₂O, together with a eukaryotic medium, wherein said ferroussulfate.EDTA and said ZnSO₄.7H₂O are each present in an amount whichsupports the high-density growth of Chinese hamster ovary cells insuspension culture.
 100. The eukaryotic cell culture medium according toclaim 99, further comprising a polyanionic or polycationic compound,wherein said polyanionic or polycationic compound is present in anamount sufficient to prevent cell clumping and/or increase the level ofrecombinant protein expression.
 101. The eukaryotic cell culture mediumaccording to claim 100, wherein said polyanionic compound is apolysulfonated compound or a polysulfated compound.
 102. The eukaryoticcell culture medium according to claim 101, wherein said polysulfonatedor polysulfated compound is selected from the group consisting ofdextran sulfate, heparin, heparan sulfate, chondroitin sulfate, dermatansulfate, pentosan sulfate and a proteoglycan.
 103. The eukaryotic cellculture medium according to claim 102, wherein said polysulfonated orpolysulfated compound is dextran sulfate.
 104. The eukaryotic cellculture medium according to claim 103, wherein said dextran sulfate hasan average molecular weight of 5,000 daltons.
 105. The eukaryotic cellculture medium obtained according to claim 99 or 100, further comprisingone or more ingredients selected from the group consisting ofL-arginine, L-asparagine.H₂O, L-aspartic acid, L-glutamic acid,L-histidine, hydroxy-L-proline, L-isoleucine, L-leucine, L-lysine.HCl,L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine, L-valine, L-cystine.2HCl, Na₂HPO₄,pyridoxine.HCl, thiamine.HCl, glutathione, cupric sulfate.5H₂O, cadmiumchloride.5H₂O, cobalt chloride.2H₂O, stannous chloride.2H₂O, manganoussulfate.H₂O, nickel sulfate.6H₂O, sodium metavanadate, ammoniummolybdate.4H₂O, barium acetate, potassium bromide, potassium iodide,chromium sulfate, sodium fluoride, silver nitrate, rubidium chloride,zirconyl chloride, aluminum. chloride, germanium dioxide, titaniumtetrachloride, sodium metasilicate, magnesium chloride (anhydrous),D-calcium pantothenate, calcium nitrate. 4H₂O, potassium chloride,ascorbic acid magnesium salt phosphate, pluronic F68 10% solution,Na₂HP0₄, D-glucose, folic acid, riboflavin, biotin, choline chloride,niacinamide, i-inositol, sodium pyruvate, vitamin B-12,β-mercaptoethanol, para-aminobenzoic acid, β-glycerophosphate, sodiumselenite, ethanolamine.HCl, monothioglycerol, and sodium bicarbonate,spermine, putrescine.2HCl, wherein each of said ingredients is presentin an amount which supports the high-density growth of Chinese hamsterovary cells in suspension culture and/or the expression of recombinantprotein. 106-128. (canceled)
 129. A kit for the cultivation of amammalian epithelial cell in suspension in vitro, said kit comprisingone or more containers, wherein a first container contains a mediumcomprising a Fe²⁺ chelate and a Zn²⁺ salt, wherein said medium iscapable of supporting the high-density growth of mammalian cells insuspension culture and/or the expression of recombinant protein.130-139. (canceled)